Oligomer-opioid agonist conjugates

ABSTRACT

The invention provides compounds that are chemically modified by covalent attachment of a water-soluble oligomer. A compound of the invention, when administered by any of a number of administration routes, exhibits characteristics that are different from those of the compound not attached to the water-soluble oligomer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application (i) is a Continuation-In-Part of International PatentApplication No. PCT/US2008/003353, which claims priority to U.S.Provisional Application Ser. No. 60/906,387, filed Mar. 12, 2007, andadditionally (ii) claims priority to U.S. Provisional Application No.61/192,261, filed Sep. 16, 2008 and to U.S. Provisional Application No.61/227,399, filed Jul. 21, 2009, the disclosures all of which areincorporated herein by reference.

FIELD

This invention provides (among other things) chemically modified opioidagonists that possess certain advantages over opioid agonists lackingthe chemical modification. The chemically modified opioid agonistsdescribed herein relate to and/or have application(s) in (among others)the fields of drug discovery, pharmacotherapy, physiology, organicchemistry and polymer chemistry.

BACKGROUND

Opioid agonists, such as morphine, have long been used to treat patientssuffering from pain. Opioid agonists exert their analgesic and otherpharmacological effects through interactions with opioid receptors, ofwhich, there are three main classes: mu (μ) receptors, kappa (κ)receptors, and delta (δ) receptors. Most of the clinically used opioidagonists are relatively selective for mu receptors, although opioidagonists typically have agonist activity at other opioid receptors(particularly at increased concentrations).

Opioids exert their effects by selectively inhibiting the release ofneurotransmitters, such as acetylcholine, norepinephrine, dopamine,serotonin, and substance P.

Pharmacologically, opioid agonists represent an important class ofagents employed in the management of pain. Unfortunately, the use ofopioid agonists is associated with the potential for abuse. In addition,oral administration of opioid agonists often results in significantfirst pass metabolism. Furthermore, administration of opioid agonistsresults in significant CNS-mediated effects, such as slowed breathing,which can result in death. Thus, a reduction of any one of these orother characteristics would enhance their desirability as therapeuticdrugs.

The present disclosure seeks to address these and other needs in the artby providing (among other things) a conjugate of a water-soluble,non-peptidic oligomer and a opioid agonist.

SUMMARY

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of an opioid agonist covalently attached(preferably via a stable linkage) to a water-soluble, non-peptidicoligomer.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of a kappa opioid agonist covalentlyattached (preferably via a stable linkage) to a water-soluble,non-peptidic oligomer [wherein it is understood that a kappa opioidagonist (i) is preferentially selective for kappa opioid receptors overboth mu opioid receptors and delta opioid receptors within the samemammalian species, and (ii) will have agonist activity at the kappareceptor].

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of a mu opioid agonist covalently attached(preferably via a stable linkage) to a water-soluble, non-peptidicoligomer [wherein it is understood that a kappa opioid agonist (i) ispreferentially selective for mu opioid receptors over both kappa opioidreceptors and delta opioid receptors within the same mammalian species,and (ii) will have agonist activity at the mu receptor].

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of an opioid agonist covalently attachedvia a stable linkage to a water-soluble, non-peptidic oligomer, whereinthe opioid agonist has a structure encompassed by the following formula:

wherein:

R¹ is H or an organic radical [such as methyl, ethyl and —C(O)CH₃];

R² is H or OH;

R³ is H or an organic radical;

R⁴ is H or an organic radical;

the dotted line (“---”) represents an optional double bond;

Y¹ is O (oxygen) or S; and

R⁵ is selected from the group consisting of

(without regard to stereochemistry), wherein R⁶ is an organic radical[including —C(O)CH₃].

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of an opioid agonist covalently attachedvia a stable or degradable linkage to a water-soluble, non-peptidicoligomer, wherein the opioid agonist is selected from the groupconsisting of asimadoline, bremazocine, enadoline, ethylketocyclazocine,GR89,696, ICI204448, ICI197067, PD117,302, nalbuphine, pentazocine,quadazocine (WIN 44,441-3), salvinorin A, spiradoline, TRK-820, U50488,and U69593.

In one or more embodiments of the invention, a composition is provided,the composition comprising:

(i) a compound comprising a residue of an opioid agonist covalentlyattached via a stable linkage to a water-soluble, non-peptidic oligomer;and

(ii) optionally, a pharmaceutically acceptable excipient.

In one or more embodiments of the invention, a dosage form is provided,the dosage form comprising a compound comprising a residue of an opioidagonist covalently attached via a stable linkage to a water-soluble,non-peptidic oligomer.

In one or more embodiments of the invention, a method is provided, themethod comprising covalently attaching a water-soluble, non-peptidicoligomer to an opioid agonist.

In one or more embodiments of the invention, a method is provided, themethod comprising administering a compound comprising a residue of anopioid agonist covalently attached via a stable linkage to awater-soluble, non-peptidic oligomer.

In one or more embodiments of the invention, a method is provided, themethod comprising binding (e.g., selectively binding) mu opioidreceptors, wherein said binding is achieved by administering a compoundcomprising a residue of an opioid agonist covalently attached to awater-soluble, non-peptidic oligomer. In one or more embodiments of theinvention, a method is provided, the method comprising binding (e.g.,selectively binding) mu opioid receptors, wherein said binding isachieved by administering an effective amount of a compound comprising aresidue of an opioid agonist covalently attached to a water-soluble,non-peptidic oligomer to a mammalian patient.

In one or more embodiments of the invention, a method is provided, themethod comprising binding (e.g., selectively binding) kappa opioidreceptors, wherein said binding is achieved by administering a compoundcomprising a residue of an opioid agonist covalently attached to awater-soluble, non-peptidic oligomer. In one or more embodiments of theinvention, a method is provided, the method comprising binding (e.g.,selectively binding) kappa opioid receptors, wherein said binding isachieved by administering an effective amount of a compound comprising aresidue of an opioid agonist covalently attached to a water-soluble,non-peptidic oligomer to a mammalian patient.

These and other objects, aspects, embodiments and features of theinvention will become more fully apparent when read in conjunction withthe following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the fold changes in binding affinity for mu,kappa, and delta receptors over parent molecule, nalbuphine, plotted asa function of PEG length for PEG-nalbuphine conjugates, as described ingreater detail in Example 4. As shown in FIG. 1, binding affinitydecreases as a function of PEG chain length at the mu and kappa opioidreceptors, but not at the delta opioid receptors, thereby demonstratingthat PEG conjugation differently affects binding at these opioidreceptor subtypes.

FIG. 2A and FIG. 2B are graphs showing the in vitro permeability andefflux ratios of various nalbuphine conjugates, as described in greaterdetail in Example 9. These graphs show that (i) the permeability ofPEG-nalbuphine conjugates in Caco-2 cells decreases as a function of PEGchain length (FIG. 2A) and, (ii) PEG-nalbuphine conjugates are likelysubstrates for efflux transporters (FIG. 2B).

FIG. 3 is a graph showing brain:plasma ratios of various PEG-nalbuphineconjugates, as described in greater detail in Example 10. This graphshows PEG conjugation results in a decrease in the brain:plasma ratiosof nalbuphine.

FIG. 4 is a graph showing percent writhing per total number of mice, n,in the study group, versus dose of mPEG_(n)-O-morphine conjugateadministered in an analgesic assay for evaluating the extent ofreduction or prevention of visceral pain in mice as described in detailin Example 18. Morphine was used as a control; unconjugated parentmolecule, morphine sulfate, was also administered to provide anadditional point of reference. Conjugates belonging to the followingconjugate series: mPEG_(2-7,9)-O-morphine were evaluated.

FIG. 5 is a graph showing percent writhing per total number of mice, n,in the study group, versus dose of mPEG_(n)-O-hydroxycodone conjugateadministered in an analgesic assay for evaluating the extent ofreduction or prevention of visceral pain in mice as described in detailin Example 18. Morphine was used as a control; unconjugated parentmolecule, oxycodone, was also administered to provide an additionalpoint of reference. Conjugates belonging to the following conjugateseries: mPEG_(1-4, 6, 7,9)-O-hydroxycodone were evaluated.

FIG. 6 is a graph showing percent writhing per total number of mice, n,in the study group, versus dose of mPEG_(n)-O-codeine conjugateadministered in an analgesic assay for evaluating the extent ofreduction or prevention of visceral pain in mice as described in detailin Example 18. Morphine was used as a control; unconjugated parentmolecule, codeine, was also administered to provide an additional pointof reference. Conjugates belonging to the following conjugate series:mPEG_(3-7,9)-O-codeine were evaluated.

FIGS. 7-9 are plots indicating the results of a hot plate latencyanalgesic assay in mice as described in detail in Example 19.Specifically, the figures correspond to graphs showing latency (time tolick hindpaw), in seconds versus dose of compound. FIG. 7 providesresults for mPEG₁₋₅-O-hydroxycodone conjugates as well as forunconjugated parent molecule; FIG. 8 provides results formPEG₁₋₅-O-morphine conjugates as well for unconjugated parent molecule;and FIG. 9 provides results for mPEG₂₋₅, 9-O-codeine conjugates as wellas for the parent molecule. The presence of an asterisk by a data pointindicates p<0.05 versus saline by ANOVA/Dunnett's.

FIG. 10 shows the mean (+SD) plasma concentration-time profiles for thecompounds, oxycodone (mPEG₀-oxycodone), mPEG₁-O-hydroxycodone,mPEG₂-O-hydroxycodone, mPEG₃-O-hydroxycodone, mPEG₄-O-hydroxycodone,mPEG₅-O-hydroxycodone, mPEG₆-O-hydroxycodone, mPEG₇-O-hydroxycodone, andmPEG₉-O-hydroxycodone, following 1.0 mg/kg intravenous administration torats as described in Example 21.

FIG. 11 shows the mean (+SD) plasma concentration-time profiles for thecompounds, oxycodone (mPEG₀-oxycodone), mPEG₁-O-hydroxycodone,mPEG₂-O-hydroxycodone, mPEG₃-O-hydroxycodone, mPEG₄-O-hydroxycodone,mPEG₅-O-hydroxycodone, mPEG₆-O-hydroxycodone, mPEG₇-O-hydroxycodone, andmPEG₉-O-hydroxycodone, following 5.0 mg/kg oral administration to ratsas described in Example 21.

FIG. 12 shows the mean (+SD) plasma concentration-time profiles for thecompounds, morphine (mPEG₀-morphine), and mPEG_(1-7, 9)-O-morphineconjugates, following 1.0 mg/kg intravenous administration to rats asdescribed in detail in Example 22.

FIG. 13 shows the mean (+SD) plasma concentration-time profiles for thecompounds, morphine (mPEG₀-morphine), and mPEG_(1-7, 9)-O-morphineconjugates, following 5.0 mg/kg oral administration to rats as describedin Example 22.

FIG. 14 shows the mean (+SD) plasma concentration-time profiles for thecompounds, codeine (mPEG₀-codeine), and mPEG_(1-7, 9)-O-codeineconjugates, following 1.0 mg/kg intravenous administration to rats asdescribed in detail in Example 23.

FIG. 15 shows the mean (+SD) plasma concentration-time profiles for thecompounds, codeine (mPEG₀-codeine), and mPEG_(1-7, 9)-O-codeineconjugates, following 5.0 mg/kg oral administration to rats as describedin Example 23.

FIGS. 16A, 16B and 16C illustrate the brain:plasma ratios of variousoligomeric mPEG_(n)-O-morphine, mPEG_(n)-O-codeine andmPEG_(n)-O-hydroxycodone conjugates, respectively, following IVadministration to rats as described in Example 26. The brain:plasmaratio of atenolol is provided in each figure as a basis for comparison.

FIGS. 17A-H illustrate brain and plasma concentrations of morphine andvarious mPEG_(n)-O-morphine conjugates over time following IVadministration to rats as described in Example 27. FIG. 17A (morphine,n=0); FIG. 17B (n=1); FIG. 17C (n=2); FIG. 17D (n=3); FIG. 17E (n=4);FIG. 17F (n=5); FIG. 17G (n=6); FIG. 17H (n=7).

FIGS. 18A-H illustrate brain and plasma concentrations of codeine andvarious mPEG_(n)-O-codeine conjugates over time following IVadministration to rats as described in Example 27. FIG. 18A (codeine,n=0); FIG. 18B (n=1); FIG. 18C (n=2); FIG. 18D (n=3); FIG. 18E (n=4);FIG. 18F (n=5); FIG. 18G (n=6); FIG. 18H (n=7).

FIGS. 19A-H illustrate brain and plasma concentrations of oxycodone andvarious mPEG_(n)-O-hydroxycodone conjugates over time following IVadministration to rats as described in Example 27. FIG. 19A (oxycodone,n=0); FIG. 19B (n=1); FIG. 19C (n=2); FIG. 19D (n=3); FIG. 19E (n=4);FIG. 19F (n=5); FIG. 19G (n=6); FIG. 19H (n=7).

DETAILED DESCRIPTION

As used in this specification, the singular forms “a,” “an,” and “the”include plural referents unless the context clearly dictates otherwise.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“Water soluble, non-peptidic oligomer” indicates an oligomer that is atleast 35% (by weight) soluble, preferably greater than 70% (by weight),and more preferably greater than 95% (by weight) soluble, in water atroom temperature. Typically, an unfiltered aqueous preparation of a“water-soluble” oligomer transmits at least 75%, more preferably atleast 95%, of the amount of light transmitted by the same solution afterfiltering. It is most preferred, however, that the water-solubleoligomer is at least 95% (by weight) soluble in water or completelysoluble in water. With respect to being “non-peptidic,” an oligomer isnon-peptidic when it has less than 35% (by weight) of amino acidresidues.

The terms “monomer,” “monomeric subunit” and “monomeric unit” are usedinterchangeably herein and refer to one of the basic structural units ofa polymer or oligomer. In the case of a homo-oligomer, a singlerepeating structural unit forms the oligomer. In the case of aco-oligomer, two or more structural units are repeated—either in apattern or randomly—to form the oligomer. Preferred oligomers used inconnection with present the invention are homo-oligomers. Thewater-soluble, non-peptidic oligomer typically comprises one or moremonomers serially attached to form a chain of monomers. The oligomer canbe formed from a single monomer type (i.e., is homo-oligomeric) or twoor three monomer types (i.e., is co-oligomeric).

An “oligomer” is a molecule possessing from about 2 to about 50monomers, preferably from about 2 to about 30 monomers. The architectureof an oligomer can vary. Specific oligomers for use in the inventioninclude those having a variety of geometries such as linear, branched,or forked, to be described in greater detail below.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Unless otherwise indicated, a“PEG oligomer” (also called an oligoethylene glycol) is one in whichsubstantially all (and more preferably all) monomeric subunits areethylene oxide subunits. The oligomer may, however, contain distinct endcapping moieties or functional groups, e.g., for conjugation. Typically,PEG oligomers for use in the present invention will comprise one of thetwo following structures: “—(CH₂CH₂O)_(n)—” or“—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whether the terminal oxygen(s)has been displaced, e.g., during a synthetic transformation. For PEGoligomers, “n” varies from about 2 to 50, preferably from about 2 toabout 30, and the terminal groups and architecture of the overall PEGcan vary. When PEG further comprises a functional group, A, for linkingto, e.g., a small molecule drug, the functional group when covalentlyattached to a PEG oligomer does not result in formation of (i) anoxygen-oxygen bond (—O—O—, a peroxide linkage), or (ii) anitrogen-oxygen bond (N—O, O—N).

An “end capping group” is generally a non-reactive carbon-containinggroup attached to a terminal oxygen of a PEG oligomer. Exemplary endcapping groups comprise a C₁₋₅ alkyl group, such as methyl, ethyl andbenzyl), as well as aryl, heteroaryl, cyclo, heterocyclo, and the like.For the purposes of the present invention, the preferred capping groupshave relatively low molecular weights such as methyl or ethyl. Theend-capping group can also comprise a detectable label. Such labelsinclude, without limitation, fluorescers, chemiluminescers, moietiesused in enzyme labeling, colorimetric labels (e.g., dyes), metal ions,and radioactive moieties.

“Branched”, in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more polymers representingdistinct “arms” that extend from a branch point.

“Forked” in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more functional groups(typically through one or more atoms) extending from a branch point.

A “branch point” refers to a bifurcation point comprising one or moreatoms at which an oligomer branches or forks from a linear structureinto one or more additional arms.

The term “reactive” or “activated” refers to a functional group thatreacts readily or at a practical rate under conventional conditions oforganic synthesis. This is in contrast to those groups that either donot react or require strong catalysts or impractical reaction conditionsin order to react (i.e., a “nonreactive” or “inert” group).

“Not readily reactive,” with reference to a functional group present ona molecule in a reaction mixture, indicates that the group remainslargely intact under conditions that are effective to produce a desiredreaction in the reaction mixture.

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group will vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof encompasses protected forms thereof.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa relatively labile bond that reacts with water (i.e., is hydrolyzed)under ordinary physiological conditions. The tendency of a bond tohydrolyze in water under ordinary physiological conditions will dependnot only on the general type of linkage connecting two central atoms butalso on the substituents attached to these central atoms. Such bonds aregenerally recognizable by those of ordinary skill in the art.Appropriate hydrolytically unstable or weak linkages include but are notlimited to carboxylate ester, phosphate ester, anhydrides, acetals,ketals, acyloxyalkyl ether, imines, orthoesters, peptides,oligonucleotides, thioesters, and carbonates.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes under ordinary physiologicalconditions.

A “stable” linkage or bond refers to a chemical moiety or bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under ordinary physiologicalconditions to any appreciable extent over an extended period of time.Examples of hydrolytically stable linkages include but are not limitedto the following: carbon-carbon bonds (e.g., in aliphatic chains),ethers, amides, urethanes, amines, and the like. Generally, a stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under ordinary physiological conditions. Hydrolysis ratesof representative chemical bonds can be found in most standard chemistrytextbooks.

In the context of describing the consistency of oligomers in a givencomposition, “substantially” or “essentially” means nearly totally orcompletely, for instance, 95% or greater, more preferably 97% orgreater, still more preferably 98% or greater, even more preferably 99%or greater, yet still more preferably 99.9% or greater, with 99.99% orgreater being most preferred of some given quantity.

“Monodisperse” refers to an oligomer composition wherein substantiallyall of the oligomers in the composition have a well-defined, singlemolecular weight and defined number of monomers, as determined bychromatography or mass spectrometry. Monodisperse oligomer compositionsare in one sense pure, that is, substantially comprising moleculeshaving a single and definable number of monomers rather than severaldifferent numbers of monomers (i.e., an oligomer composition havingthree or more different oligomer sizes). A monodisperse oligomercomposition possesses a MW/Mn value of 1.0005 or less, and morepreferably, a MW/Mn value of 1.0000. By extension, a compositioncomprised of monodisperse conjugates means that substantially alloligomers of all conjugates in the composition have a single anddefinable number (as a whole number) of monomers rather than adistribution and would possess a MW/Mn value of 1.0005, and morepreferably, a MW/Mn value of 1.0000 if the oligomer were not attached tothe residue of the opioid agonist. A composition comprised ofmonodisperse conjugates can include, however, one or more nonconjugatesubstances such as solvents, reagents, excipients, and so forth.

“Bimodal,” in reference to an oligomer composition, refers to anoligomer composition wherein substantially all oligomers in thecomposition have one of two definable and different numbers (as wholenumbers) of monomers rather than a distribution, and whose distributionof molecular weights, when plotted as a number fraction versus molecularweight, appears as two separate identifiable peaks. Preferably, for abimodal oligomer composition as described herein, each peak is generallysymmetric about its mean, although the size of the two peaks may differ.Ideally, the polydispersity index of each peak in the bimodaldistribution, Mw/Mn, is 1.01 or less, more preferably 1.001 or less, andeven more preferably 1.0005 or less, and most preferably a MW/Mn valueof 1.0000. By extension, a composition comprised of bimodal conjugatesmeans that substantially all oligomers of all conjugates in thecomposition have one of two definable and different numbers (as wholenumbers) of monomers rather than a large distribution and would possessa MW/Mn value of 1.01 or less, more preferably 1.001 or less and evenmore preferably 1.0005 or less, and most preferably a MW/Mn value of1.0000 if the oligomer were not attached to the residue of the opioidagonist. A composition comprised of bimodal conjugates can include,however, one or more nonconjugate substances such as solvents, reagents,excipients, and so forth.

An “opioid agonist” is broadly used herein to refer to an organic,inorganic, or organometallic compound typically having a molecularweight of less than about 1000 Daltons (and typically less than 500Daltons) and having some degree of activity as a mu and/or kappaagonist. Opioid agonists encompass oligopeptides and other biomoleculeshaving a molecular weight of less than about 1000.

A “biological membrane” is any membrane, typically made from specializedcells or tissues, that serves as a barrier to at least some foreignentities or otherwise undesirable materials. As used herein a“biological membrane” includes those membranes that are associated withphysiological protective barriers including, for example: theblood-brain barrier (BBB); the blood-cerebrospinal fluid barrier; theblood-placental barrier; the blood-milk barrier; the blood-testesbarrier; and mucosal barriers including the vaginal mucosa, urethralmucosa, anal mucosa, buccal mucosa, sublingual mucosa, rectal mucosa,and so forth. Unless the context clearly dictates otherwise, the term“biological membrane” does not include those membranes associated withthe middle gastro-intestinal tract (e.g., stomach and small intestines).

A “biological membrane crossing rate,” as used herein, provides ameasure of a compound's ability to cross a biological membrane (such asthe membrane associated with the blood-brain barrier). A variety ofmethods can be used to assess transport of a molecule across any givenbiological membrane. Methods to assess the biological membrane crossingrate associated with any given biological barrier (e.g., theblood-cerebrospinal fluid barrier, the blood-placental barrier, theblood-milk barrier, the intestinal barrier, and so forth), are known inthe art, described herein and/or in the relevant literature, and/or canbe determined by one of ordinary skill in the art.

A “reduced rate of metabolism” in reference to the present invention,refers to a measurable reduction in the rate of metabolism of awater-soluble oligomer-small molecule drug conjugate as compared to rateof metabolism of the small molecule drug not attached to thewater-soluble oligomer (i.e., the small molecule drug itself) or areference standard material. In the special case of “reduced first passrate of metabolism,” the same “reduced rate of metabolism” is requiredexcept that the small molecule drug (or reference standard material) andthe corresponding conjugate are administered orally. Orally administereddrugs are absorbed from the gastro-intestinal tract into the portalcirculation and must pass through the liver prior to reaching thesystemic circulation. Because the liver is the primary site of drugmetabolism or biotransformation, a substantial amount of drug can bemetabolized before it ever reaches the systemic circulation. The degreeof first pass metabolism, and thus, any reduction thereof, can bemeasured by a number of different approaches. For instance, animal bloodsamples can be collected at timed intervals and the plasma or serumanalyzed by liquid chromatography/mass spectrometry for metabolitelevels. Other techniques for measuring a “reduced rate of metabolism”associated with the first pass metabolism and other metabolic processesare known in the art, described herein and/or in the relevantliterature, and/or can be determined by one of ordinary skill in theart. Preferably, a conjugate of the invention can provide a reduced rateof metabolism reduction satisfying at least one of the following values:at least about 30%; at least about 40%; at least about 50%; at leastabout 60%; at least about 70%; at least about 80%; and at least about90%. A compound (such as a small molecule drug or conjugate thereof)that is “orally bioavailable” is one that preferably possesses abioavailability when administered orally of greater than 25%, andpreferably greater than 70%, where a compound's bioavailability is thefraction of administered drug that reaches the systemic circulation inunmetabolized form.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to20 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includemethyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl when three or more carbon atoms are referenced. An “alkenyl”group is an alkyl of 2 to 20 carbon atoms with at least onecarbon-carbon double bond.

The terms “substituted alkyl” or “substituted C_(q-r) alkyl” where q andr are integers identifying the range of carbon atoms contained in thealkyl group, denotes the above alkyl groups that are substituted by one,two or three halo (e.g., F, Cl, Br, I), trifluoromethyl, hydroxy, C₁₋₇alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, and soforth), C₁₋₇ alkoxy, C₁₋₇ acyloxy, C₃₋₇ heterocyclic, amino, phenoxy,nitro, carboxy, carboxy, acyl, cyano. The substituted alkyl groups maybe substituted once, twice or three times with the same or withdifferent substituents.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl. “Lower alkenyl” refers to a loweralkyl group of 2 to 6 carbon atoms having at least one carbon-carbondouble bond.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy,benzyl, etc.), preferably C₁-C₇.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to component that can be included in the compositions ofthe invention in order to provide for a composition that has anadvantage (e.g., more suited for administration to a patient) over acomposition lacking the component and that is recognized as not causingsignificant adverse toxicological effects to a patient.

The term “aryl” means an aromatic group having up to 14 carbon atoms.Aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl,naphthacenyl, and the like. “Substituted phenyl” and “substituted aryl”denote a phenyl group and aryl group, respectively, substituted withone, two, three, four or five (e.g. 1-2, 1-3 or 1-4 substituents) chosenfrom halo (F, Cl, Br, I), hydroxy, hydroxy, cyano, nitro, alkyl (e.g.,C₁₋₆ alkyl), alkoxy (e.g., C₁₋₆ alkoxy), benzyloxy, carboxy, aryl, andso forth.

An “aromatic-containing moiety” is a collection of atoms containing atleast aryl and optionally one or more atoms. Suitablearomatic-containing moieties are described herein.

For simplicity, chemical moieties are defined and referred to throughoutprimarily as univalent chemical moieties (e.g., alkyl, aryl, etc.).Nevertheless, such terms are also used to convey correspondingmultivalent moieties under the appropriate structural circumstancesclear to those skilled in the art. For example, while an “alkyl” moietygenerally refers to a monovalent radical (e.g., CH₃—CH₂—), in certaincircumstances a bivalent linking moiety can be “alkyl,” in which casethose skilled in the art will understand the alkyl to be a divalentradical (e.g., —CH₂—CH₂—), which is equivalent to the term “alkylene.”(Similarly, in circumstances in which a divalent moiety is required andis stated as being “aryl,” those skilled in the art will understand thatthe term “aryl” refers to the corresponding divalent moiety, arylene).All atoms are understood to have their normal number of valences forbond formation (i.e., 4 for carbon, 3 for N, 2 for O, and 2, 4, or 6 forS, depending on the oxidation state of the S).

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a water-soluble oligomer-small moleculedrug conjugate present in a composition that is needed to provide athreshold level of active agent and/or conjugate in the bloodstream orin the target tissue. The precise amount will depend upon numerousfactors, e.g., the particular active agent, the components and physicalcharacteristics of the composition, intended patient population, patientconsiderations, and the like, and can readily be determined by oneskilled in the art, based upon the information provided herein andavailable in the relevant literature.

A “difunctional” oligomer is an oligomer having two functional groupscontained therein, typically at its termini. When the functional groupsare the same, the oligomer is said to be homodifunctional. When thefunctional groups are different, the oligomer is said to beheterobifunctional.

A basic reactant or an acidic reactant described herein include neutral,charged, and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aconjugate as described herein, typically, but not necessarily, in theform of a water-soluble oligomer-small molecule drug conjugate, andincludes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may but need not necessarily occur, so that the descriptionincludes instances where the circumstance occurs and instances where itdoes not.

As indicated above, the present invention is directed to (among otherthings) a compound comprising a residue of an opioid agonist covalentlyattached via a stable or degradable linkage to a water-soluble,non-peptidic oligomer.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of an opioid agonist covalently attachedvia a stable or degradable linkage to a water-soluble, non-peptidicoligomer, wherein the opioid agonist has a structure encompassed by thefollowing formula:

wherein:

R¹ is H or an organic radical [such as methyl, ethyl and —C(O)CH₃];

R² is H or OH;

R³ is H or an organic radical;

R⁴ is H or an organic radical;

the dotted line (“---”) represents an optional double bond;

Y¹ is O or S; and

R⁵ is selected from the group consisting of

(without regard to stereochemistry), wherein R⁶ is an organic radical[including C(O)CH₃]. Exemplary R³ groups include lower alkyl such asmethyl, ethyl, isopropyl, and the like, as well as the following:

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of an opioid agonist covalently attachedvia a stable or degradable linkage to a water-soluble, non-peptidicoligomer, wherein the opioid agonist has a structure encompassed by thefollowing formula:

wherein:

N* is nitrogen;

Ar is selected from the group consisting of cyclohexyl, phenyl,halophenyl, methoxyphenyl, aminophenyl, pyridyl, furyl and thienyl;

Alk is selected from the group consisting of ethylene and propylene;

R_(II) is selected from the group consisting of lower alkyl, loweralkoxy, dimethylamino, cyclopropyl, 1-pyrrolidyl, morpholino (preferablylower alkyl such as ethyl);

R_(II)′ is selected from the group consisting of hydrogen, methyl andmethoxy; and

R_(II)″ is selected from the group consisting of hydrogen and an organicradical (preferably lower alkyl).

With respect to Formula II, it will be understood that, depending on theconditions, one or both of the amines—but more typically, the aminemarked with an asterisk (“N*”) in Formula II—can be protonated.

Examples of specific opioid agonists include those selected from thegroup consisting acetorphine, acetyldihydrocodeine,acetyldihydrocodeinone, acetylmorphinone, alfentanil, allylprodine,alphaprodine, anileridine, benzylmorphine, bezitramide, buprenorphine,butorphanol, clonitazene, codeine, desomorphine, dextromoramide,dezocine, diampromide, diamorphone, dihydrocodeine, dihydromorphine,dimenoxadol, dimepheptanol, dimethylthiambutene, dioxaphetyl butyrate,dipipanone, eptazocine, ethoheptazine, ethylmethylthiambutene,ethylmorphine, etonitazene, etorphine, dihydroetorphine, fentanyl andderivatives, heroin, hydrocodone, hydroxycodone, hydromorphone,hydroxypethidine, isomethadone, ketobemidone, levorphanol,levophenacylmorphan, lofentanil, meperidine, meptazinol, metazocine,methadone, metopon, morphine, myrophine, narceine, nicomorphine,norlevorphanol, normethadone, nalorphine, nalbuphine, normorphine,norpipanone, opium, oxycodone, oxymorphone, papaveretum, pentazocine,phenadoxone, phenomorphan, phenazocine, phenoperidine, piminodine,piritramide, propheptazine, promedol, properidine, propoxyphene,sufentanil, tilidine, and tramadol. In certain embodiments, the opioidagonist is selected from the group consisting of hydrocodone, morphine,hydromorphone, oxycodone, codeine, levorphanol, meperidine, methadone,oxymorphone, buprenorphine, fentanyl, dipipanone, heroin, tramadol,nalbuphine, etorphine, dihydroetorphine, butorphanol, levorphanol.

It is believed that an advantage of the compounds of the presentinvention is their ability to retain some degree of opioid agonistactivity while also exhibiting a decrease in metabolism and/or resultingin a decrease of CNS-mediated effects associated with the correspondingopioid agonist in unconjugated form. Although not wishing to be bound bytheory, it is believed that the oligomer-containing conjugates describedherein—in contrast to the unconjugated “original” opioid agonist—are notmetabolized as readily because the oligomer serves to reduce the overallaffinity of the compound to substrates that can metabolize opioidagonists. In addition (and again, not wishing to be bound by theory),the extra size introduced by the oligomer—in contrast to theunconjugated “original” opioid agonist—reduces the ability of thecompound to cross the blood-brain barrier.

Use of oligomers (e.g., from a monodisperse or bimodal composition ofoligomers, in contrast to relatively impure compositions) to form theconjugates of the invention can advantageously alter certain propertiesassociated with the corresponding small molecule drug. For instance, aconjugate of the invention, when administered by any of a number ofsuitable administration routes, such as parenteral, oral, transdermal,buccal, pulmonary, or nasal, exhibits reduced penetration across theblood-brain barrier. It is preferred that the conjugate exhibit slowed,minimal or effectively no crossing of the blood-brain barrier, whilestill crossing the gastro-intestinal (GI) walls and into the systemiccirculation if oral delivery is intended. Moreover, the conjugates ofthe invention maintain a degree of bioactivity as well asbioavailability in their conjugated form in comparison to thebioactivity and bioavailability of the compound free of all oligomers.

With respect to the blood-brain barrier (“BBB”), this barrier restrictsthe transport of drugs from the blood to the brain. This barrierconsists of a continuous layer of unique endothelial cells joined bytight junctions. The cerebral capillaries, which comprise more than 95%of the total surface area of the BBB, represent the principal route forthe entry of most solutes and drugs into the central nervous system.

For compounds whose degree of blood-brain barrier crossing ability isnot readily known, such ability can be determined using a suitableanimal model such as an in situ rat brain perfusion (“RBP”) model asdescribed herein. Briefly, the RBP technique involves cannulation of thecarotid artery followed by perfusion with a compound solution undercontrolled conditions, followed by a wash out phase to remove compoundremaining in the vascular space. (Such analyses can be conducted, forexample, by contract research organizations such as Absorption Systems,Exton, Pa.). More specifically, in the RBP model, a cannula is placed inthe left carotid artery and the side branches are tied off. Aphysiologic buffer containing the analyte (typically but not necessarilyat a 5 micromolar concentration level) is perfused at a flow rate ofabout 10 mL/minute in a single pass perfusion experiment. After 30seconds, the perfusion is stopped and the brain vascular contents arewashed out with compound-free buffer for an additional 30 seconds. Thebrain tissue is then removed and analyzed for compound concentrationsvia liquid chromatograph with tandem mass spectrometry detection(LC/MS/MS). Alternatively, blood-brain barrier permeability can beestimated based upon a calculation of the compound's molecular polarsurface area (“PSA”), which is defined as the sum of surfacecontributions of polar atoms (usually oxygens, nitrogens and attachedhydrogens) in a molecule. The PSA has been shown to correlate withcompound transport properties such as blood-brain barrier transport.Methods for determining a compound's PSA can be found, e.g., in, Ertl,P., et al., J. Med. Chem. 2000, 43, 3714-3717; and Kelder, J., et al.,Pharm. Res. 1999, 16, 1514-1519.

With respect to the blood-brain barrier, the water-soluble, non-peptidicoligomer-small molecule drug conjugate exhibits a blood-brain barriercrossing rate that is reduced as compared to the crossing rate of thesmall molecule drug not attached to the water-soluble, non-peptidicoligomer. Preferred exemplary reductions in blood-brain barrier crossingrates for the compounds described herein include reductions of: at leastabout 30%; at least about 40%; at least about 50%; at least about 60%;at least about 70%; at least about 80%; or at least about 90%, whencompared to the blood-brain barrier crossing rate of the small moleculedrug not attached to the water-soluble oligomer. A preferred reductionin the blood-brain barrier crossing rate for a conjugate is at leastabout 20%.

As indicated above, the compounds of the invention include a residue ofan opioid agonist. Assays for determining whether a given compound(regardless of whether the compound is in conjugated form or not) canact as an agonist on a mu receptor or a kappa receptors are describedinfra.

In some instances, opioid agonists can be obtained from commercialsources. In addition, opioid agonists can be obtained through chemicalsynthesis. Synthetic approaches for preparing opioid agonists aredescribed in the literature and in, for example, U.S. Pat. Nos.2,628,962, 2,654,756, 2,649,454, and 2,806,033.

Each of these (and other) opioid agonists can be covalently attached(either directly or through one or more atoms) to a water-soluble,non-peptidic oligomer.

Small molecule drugs useful in the invention generally have a molecularweight of less than 1000 Da. Exemplary molecular weights of smallmolecule drugs include molecular weights of: less than about 950; lessthan about 900; less than about 850; less than about 800; less thanabout 750; less than about 700; less than about 650; less than about600; less than about 550; less than about 500; less than about 450; lessthan about 400; less than about 350; and less than about 300.

The small molecule drug used in the invention, if chiral, may be in aracemic mixture, or an optically active form, for example, a singleoptically active enantiomer, or any combination or ratio of enantiomers(i.e., scalemic mixture). In addition, the small molecule drug maypossess one or more geometric isomers. With respect to geometricisomers, a composition can comprise a single geometric isomer or amixture of two or more geometric isomers. A small molecule drug for usein the present invention can be in its customary active form, or maypossess some degree of modification. For example, a small molecule drugmay have a targeting agent, tag, or transporter attached thereto, priorto or after covalent attachment of an oligomer. Alternatively, the smallmolecule drug may possess a lipophilic moiety attached thereto, such asa phospholipid (e.g., distearoylphosphatidylethanolamine or “DSPE,”dipalmitoylphosphatidylethanolamine or “DPPE,” and so forth) or a smallfatty acid. In some instances, however, it is preferred that the smallmolecule drug moiety does not include attachment to a lipophilic moiety.

The opioid agonist for coupling to a water-soluble, non-peptidicoligomer possesses a free hydroxyl, carboxyl, thio, amino group, or thelike (i.e., “handle”) suitable for covalent attachment to the oligomer.In addition, the opioid agonist can be modified by introduction of areactive group, preferably by conversion of one of its existingfunctional groups to a functional group suitable for formation of astable covalent linkage between the oligomer and the drug.

Accordingly, each oligomer is composed of up to three different monomertypes selected from the group consisting of: alkylene oxide, such asethylene oxide or propylene oxide; olefinic alcohol, such as vinylalcohol, 1-propenol or 2-propenol; vinyl pyrrolidone; hydroxyalkylmethacrylamide or hydroxyalkyl methacrylate, where alkyl is preferablymethyl; α-hydroxy acid, such as lactic acid or glycolic acid;phosphazene, oxazoline, amino acids, carbohydrates such asmonosaccharides, saccharide or mannitol; and N-acryloylmorpholine.Preferred monomer types include alkylene oxide, olefinic alcohol,hydroxyalkyl methacrylamide or methacrylate, N-acryloylmorpholine, andα-hydroxy acid. Preferably, each oligomer is, independently, aco-oligomer of two monomer types selected from this group, or, morepreferably, is a homo-oligomer of one monomer type selected from thisgroup.

The two monomer types in a co-oligomer may be of the same monomer type,for example, two alkylene oxides, such as ethylene oxide and propyleneoxide. Preferably, the oligomer is a homo-oligomer of ethylene oxide.Usually, although not necessarily, the terminus (or termini) of theoligomer that is not covalently attached to a small molecule is cappedto render it unreactive. Alternatively, the terminus may include areactive group. When the terminus is a reactive group, the reactivegroup is either selected such that it is unreactive under the conditionsof formation of the final oligomer or during covalent attachment of theoligomer to a small molecule drug, or it is protected as necessary. Onecommon end-functional group is hydroxyl or —OH, particularly foroligoethylene oxides.

The water-soluble, non-peptidic oligomer (e.g., “POLY” in variousstructures provided herein) can have any of a number of differentgeometries. For example, it can be linear, branched, or forked. Mosttypically, the water-soluble, non-peptidic oligomer is linear or isbranched, for example, having one branch point. Although much of thediscussion herein is focused upon poly(ethylene oxide) as anillustrative oligomer, the discussion and structures presented hereincan be readily extended to encompass any of the water-soluble,non-peptidic oligomers described above.

The molecular weight of the water-soluble, non-peptidic oligomer,excluding the linker portion, is generally relatively low. Exemplaryvalues of the molecular weight of the water-soluble polymer include:below about 1500; below about 1450; below about 1400; below about 1350;below about 1300; below about 1250; below about 1200; below about 1150;below about 1100; below about 1050; below about 1000; below about 950;below about 900; below about 850; below about 800; below about 750;below about 700; below about 650; below about 600; below about 550;below about 500; below about 450; below about 400; below about 350;below about 300; below about 250; below about 200; and below about 100Daltons.

Exemplary ranges of molecular weights of the water-soluble, non-peptidicoligomer (excluding the linker) include: from about 100 to about 1400Daltons; from about 100 to about 1200 Daltons; from about 100 to about800 Daltons; from about 100 to about 500 Daltons; from about 100 toabout 400 Daltons; from about 200 to about 500 Daltons; from about 200to about 400 Daltons; from about 75 to 1000 Daltons; and from about 75to about 750 Daltons.

Preferably, the number of monomers in the water-soluble, non-peptidicoligomer falls within one or more of the following ranges: between about1 and about 30 (inclusive); between about 1 and about 25; between about1 and about 20; between about 1 and about 15; between about 1 and about12; between about 1 and about 10. In certain instances, the number ofmonomers in series in the oligomer (and the corresponding conjugate) isone of 1, 2, 3, 4, 5, 6, 7, or 8. In additional embodiments, theoligomer (and the corresponding conjugate) contains 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 monomers. In yet further embodiments, theoligomer (and the corresponding conjugate) possesses 21, 22, 23, 24, 25,26, 27, 28, 29 or 30 monomers in series. Thus, for example, when thewater-soluble, non-peptidic oligomer includes CH₃—(OCH₂CH₂)_(n)—, “n” isan integer that can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, andcan fall within one or more of the following ranges: between about 1 andabout 25; between about 1 and about 20; between about 1 and about 15;between about 1 and about 12; between about 1 and about 10.

When the water-soluble, non-peptidic oligomer has 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 monomers, these values correspond to a methoxy end-cappedoligo(ethylene oxide) having a molecular weights of about 75, 119, 163,207, 251, 295, 339, 383, 427, and 471 Daltons, respectively. When theoligomer has 11, 12, 13, 14, or 15 monomers, these values correspond tomethoxy end-capped oligo(ethylene oxide) having molecular weightscorresponding to about 515, 559, 603, 647, and 691 Daltons,respectively.

When the water-soluble, non-peptidic oligomer is attached to the opioidagonist (in contrast to the step-wise addition of one or more monomersto effectively “grow” the oligomer onto the opioid agonist), it ispreferred that the composition containing an activated form of thewater-soluble, non-peptidic oligomer be monodispersed. In thoseinstances, however, where a bimodal composition is employed, thecomposition will possess a bimodal distribution centering around any twoof the above numbers of monomers. Ideally, the polydispersity index ofeach peak in the bimodal distribution, Mw/Mn, is 1.01 or less, and evenmore preferably, is 1.001 or less, and even more preferably is 1.0005 orless. Most preferably, each peak possesses a MW/Mn value of 1.0000. Forinstance, a bimodal oligomer may have any one of the following exemplarycombinations of monomer subunits: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, and so forth; 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, and soforth; 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, and so forth; 4-5, 4-6, 4-7,4-8, 4-9, 4-10, and so forth; 5-6, 5-7, 5-8, 5-9, 5-10, and so forth;6-7, 6-8, 6-9, 6-10, and so forth; 7-8, 7-9, 7-10, and so forth; and8-9, 8-10, and so forth.

In some instances, the composition containing an activated form of thewater-soluble, non-peptidic oligomer will be trimodal or eventetramodal, possessing a range of monomers units as previouslydescribed. Oligomer compositions possessing a well-defined mixture ofoligomers (i.e., being bimodal, trimodal, tetramodal, and so forth) canbe prepared by mixing purified monodisperse oligomers to obtain adesired profile of oligomers (a mixture of two oligomers differing onlyin the number of monomers is bimodal; a mixture of three oligomersdiffering only in the number of monomers is trimodal; a mixture of fouroligomers differing only in the number of monomers is tetramodal), oralternatively, can be obtained from column chromatography of apolydisperse oligomer by recovering the “center cut”, to obtain amixture of oligomers in a desired and defined molecular weight range.

It is preferred that the water-soluble, non-peptidic oligomer isobtained from a composition that is preferably unimolecular ormonodisperse. That is, the oligomers in the composition possess the samediscrete molecular weight value rather than a distribution of molecularweights. Some monodisperse oligomers can be purchased from commercialsources such as those available from Sigma-Aldrich, or alternatively,can be prepared directly from commercially available starting materialssuch as Sigma-Aldrich. Water-soluble, non-peptidic oligomers can beprepared as described in Chen Y., Baker, G. L., J. Org. Chem., 6870-6873(1999), WO 02/098949, and U.S. Patent Application Publication2005/0136031.

When present, the spacer moiety (through which the water-soluble,non-peptidic polymer is attached to the opioid agonist) may be a singlebond, a single atom, such as an oxygen atom or a sulfur atom, two atoms,or a number of atoms. A spacer moiety is typically but is notnecessarily linear in nature. The spacer moiety, “X” is preferablyhydrolytically stable, and is preferably also enzymatically stable.Preferably, the spacer moiety “X” is one having a chain length of lessthan about 12 atoms, and preferably less than about 10 atoms, and evenmore preferably less than about 8 atoms and even more preferably lessthan about 5 atoms, whereby length is meant the number of atoms in asingle chain, not counting substituents. For instance, a urea linkagesuch as this, R_(oligomer)—NH—(C═O)—NH—R′_(drug), is considered to havea chain length of 3 atoms (—NH—C(O)—NH—). In selected embodiments, thespacer moiety linkage does not comprise further spacer groups.

In some instances, the spacer moiety “X” comprises an ether, amide,urethane, amine, thioether, urea, or a carbon-carbon bond. Functionalgroups such as those discussed below, and illustrated in the examples,are typically used for forming the linkages. The spacer moiety may lesspreferably also comprise (or be adjacent to or flanked by) spacergroups, as described further below.

More specifically, in selected embodiments, a spacer moiety, X, may beany of the following: “—” (i.e., a covalent bond, that may be stable ordegradable, between the residue of the small molecule opioid agonist andthe water-soluble, non-peptidic oligomer), —C(O)O—, —OC(O)—,—CH₂—C(O)O—, —CH₂—OC(O)—, —C(O)O—CH₂—, —OC(O)—CH₂, —O—, —NH—, —S—,—C(O)—, C(O)—NH, NH—C(O)—NH, O—C(O)—NH, —C(S)—, —CH₂—, —CH₂—CH₂—,—CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—,—CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂, —CH₂—CH₂—NH—C(O)—CH₂—CH₂, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, bivalent cycloalkyl group,—N(R⁶)—, R⁶ is H or an organic radical selected from the groupconsisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl and substituted aryl.

For purposes of the present invention, however, a group of atoms is notconsidered a spacer moiety when it is immediately adjacent to anoligomer segment, and the group of atoms is the same as a monomer of theoligomer such that the group would represent a mere extension of theoligomer chain.

The linkage “X” between the water-soluble, non-peptidic oligomer and thesmall molecule is typically formed by reaction of a functional group ona terminus of the oligomer (or one or more monomers when it is desiredto “grow” the oligomer onto the opioid agonist) with a correspondingfunctional group within the opioid agonist. Illustrative reactions aredescribed briefly below. For example, an amino group on an oligomer maybe reacted with a carboxylic acid or an activated carboxylic acidderivative on the small molecule, or vice versa, to produce an amidelinkage. Alternatively, reaction of an amine on an oligomer with anactivated carbonate (e.g. succinimidyl or benzotriazyl carbonate) on thedrug, or vice versa, forms a carbamate linkage. Reaction of an amine onan oligomer with an isocyanate (R—N═C═O) on a drug, or vice versa, formsa urea linkage (R—NH—(C═O)—NH—R′). Further, reaction of an alcohol(alkoxide) group on an oligomer with an alkyl halide, or halide groupwithin a drug, or vice versa, forms an ether linkage. In yet anothercoupling approach, a small molecule having an aldehyde function iscoupled to an oligomer amino group by reductive amination, resulting information of a secondary amine linkage between the oligomer and thesmall molecule.

A particularly preferred water-soluble, non-peptidic oligomer is anoligomer bearing an aldehyde functional group. In this regard, theoligomer will have the following structure:CH₃O—(CH₂—CH₂—O)_(n)—(CH₂)_(p)—C(O)H, wherein (n) is one of 1, 2, 3, 4,5, 6, 7, 8, 9 and 10 and (p) is one of 1, 2, 3, 4, 5, 6 and 7. Preferred(n) values include 3, 5 and 7 and preferred (p) values 2, 3 and 4. Inaddition, the carbon atom alpha to the —C(O)H moiety can optionally besubstituted with alkyl.

Typically, the terminus of the water-soluble, non-peptidic oligomer notbearing a functional group is capped to render it unreactive. When theoligomer does include a further functional group at a terminus otherthan that intended for formation of a conjugate, that group is eitherselected such that it is unreactive under the conditions of formation ofthe linkage “X,” or it is protected during the formation of the linkage“X.”

As stated above, the water-soluble, non-peptidic oligomer includes atleast one functional group prior to conjugation. The functional grouptypically comprises an electrophilic or nucleophilic group for covalentattachment to a small molecule, depending upon the reactive groupcontained within or introduced into the small molecule. Examples ofnucleophilic groups that may be present in either the oligomer or thesmall molecule include hydroxyl, amine, hydrazine (—NHNH₂), hydrazide(—C(O)NHNH₂), and thiol. Preferred nucleophiles include amine,hydrazine, hydrazide, and thiol, particularly amine. Most small moleculedrugs for covalent attachment to an oligomer will possess a freehydroxyl, amino, thio, aldehyde, ketone, or carboxyl group.

Examples of electrophilic functional groups that may be present ineither the oligomer or the small molecule include carboxylic acid,carboxylic ester, particularly imide esters, orthoester, carbonate,isocyanate, isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate,methacrylate, acrylamide, sulfone, maleimide, disulfide, iodo, epoxy,sulfonate, thiosulfonate, silane, alkoxysilane, and halosilane. Morespecific examples of these groups include succinimidyl ester orcarbonate, imidazoyl ester or carbonate, benzotriazole ester orcarbonate, vinyl sulfone, chloroethylsulfone, vinylpyridine, pyridyldisulfide, iodoacetamide, glyoxal, dione, mesylate, tosylate, andtresylate (2,2,2-trifluoroethanesulfonate).

Also included are sulfur analogs of several of these groups, such asthione, thione hydrate, thioketal, is 2-thiazolidine thione, etc., aswell as hydrates or protected derivatives of any of the above moieties(e.g. aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal,ketal, thioketal, thioacetal).

An “activated derivative” of a carboxylic acid refers to a carboxylicacid derivative which reacts readily with nucleophiles, generally muchmore readily than the underivatized carboxylic acid. Activatedcarboxylic acids include, for example, acid halides (such as acidchlorides), anhydrides, carbonates, and esters. Such esters includeimide esters, of the general form —(CO)O—N[(CO)—]₂; for example,N-hydroxysuccinimidyl (NHS) esters or N-hydroxyphthalimidyl esters. Alsopreferred are imidazolyl esters and benzotriazole esters. Particularlypreferred are activated propionic acid or butanoic acid esters, asdescribed in co-owned U.S. Pat. No. 5,672,662. These include groups ofthe form —(CH₂)₂₋₃C(═O)O-Q, where Q is preferably selected fromN-succinimide, N-sulfosuccinimide, N-phthalimide, N-glutarimide,N-tetrahydrophthalimide, N-norbornene-2,3-dicarboximide, benzotriazole,7-azabenzotriazole, and imidazole.

Other preferred electrophilic groups include succinimidyl carbonate,maleimide, benzotriazole carbonate, glycidyl ether, imidazoyl carbonate,p-nitrophenyl carbonate, acrylate, tresylate, aldehyde, and orthopyridyldisulfide.

These electrophilic groups are subject to reaction with nucleophiles,e.g. hydroxy, thio, or amino groups, to produce various bond types.Preferred for the present invention are reactions which favor formationof a hydrolytically stable linkage. For example, carboxylic acids andactivated derivatives thereof, which include orthoesters, succinimidylesters, imidazolyl esters, and benzotriazole esters, react with theabove types of nucleophiles to form esters, thioesters, and amides,respectively, of which amides are the most hydrolytically stable.Carbonates, including succinimidyl, imidazolyl, and benzotriazolecarbonates, react with amino groups to form carbamates. Isocyanates(R—N═C═O) react with hydroxyl or amino groups to form, respectively,carbamate (RNH—C(O)—OR′) or urea (RNH—C(O)—NHR′) linkages. Aldehydes,ketones, glyoxals, diones and their hydrates or alcohol adducts (i.e.aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, andketal) are preferably reacted with amines, followed by reduction of theresulting imine, if desired, to provide an amine linkage (reductiveamination).

Several of the electrophilic functional groups include electrophilicdouble bonds to which nucleophilic groups, such as thiols, can be added,to form, for example, thioether bonds. These groups include maleimides,vinyl sulfones, vinyl pyridine, acrylates, methacrylates, andacrylamides. Other groups comprise leaving groups that can be displacedby a nucleophile; these include chloroethyl sulfone, pyridyl disulfides(which include a cleavable S—S bond), iodoacetamide, mesylate, tosylate,thiosulfonate, and tresylate. Epoxides react by ring opening by anucleophile, to form, for example, an ether or amine bond. Reactionsinvolving complementary reactive groups such as those noted above on theoligomer and the small molecule are utilized to prepare the conjugatesof the invention.

In some instances the opioid agonist may not have a functional groupsuited for conjugation. In this instance, it is possible to modify the“original” opioid agonist so that it does have the desired functionalgroup. For example, if the opioid agonist has an amide group, but anamine group is desired, it is possible to modify the amide group to anamine group by way of a Hofmann rearrangement, Curtius rearrangement(once the amide is converted to an azide) or Lossen rearrangement (onceamide is concerted to hydroxamide followed by treatment withtolyene-2-sulfonyl chloride/base).

It is possible to prepare a conjugate of small molecule opioid agonistbearing a carboxyl group wherein the carboxyl group-bearing smallmolecule opioid agonist is coupled to an amino-terminated oligomericethylene glycol, to provide a conjugate having an amide group covalentlylinking the small molecule opioid agonist to the oligomer. This can beperformed, for example, by combining the carboxyl group-bearing smallmolecule opioid agonist with the amino-terminated oligomeric ethyleneglycol in the presence of a coupling reagent, (such asdicyclohexylcarbodiimide or “DCC”) in an anhydrous organic solvent.

Further, it is possible to prepare a conjugate of a small moleculeopioid agonist bearing a hydroxyl group wherein the hydroxylgroup-bearing small molecule opioid agonist is coupled to an oligomericethylene glycol halide to result in an ether (—O—) linked small moleculeconjugate. This can be performed, for example, by using sodium hydrideto deprotonate the hydroxyl group followed by reaction with ahalide-terminated oligomeric ethylene glycol.

In another example, it is possible to prepare a conjugate of a smallmolecule opioid agonist bearing a ketone group by first reducing theketone group to form the corresponding hydroxyl group. Thereafter, thesmall molecule opioid agonist now bearing a hydroxyl group can becoupled as described herein.

In still another instance, it is possible to prepare a conjugate of asmall molecule opioid agonist bearing an amine group. In one approach,the amine group-bearing small molecule opioid agonist and analdehyde-bearing oligomer are dissolved in a suitable buffer after whicha suitable reducing agent (e.g., NaCNBH₃) is added. Following reduction,the result is an amine linkage formed between the amine group of theamine group-containing small molecule opioid agonist and the carbonylcarbon of the aldehyde-bearing oligomer.

In another approach for preparing a conjugate of a small molecule opioidagonist bearing an amine group, a carboxylic acid-bearing oligomer andthe amine group-bearing small molecule opioid agonist are combined,typically in the presence of a coupling reagent (e.g., DCC). The resultis an amide linkage formed between the amine group of the aminegroup-containing small molecule opioid agonist and the carbonyl of thecarboxylic acid-bearing oligomer.

Exemplary conjugates of the opioid agonists of Formula I include thosehaving the following structure:

wherein each of R², R³, R⁴, the dotted line (“---”), Y¹ and R⁵ is aspreviously defined with respect to Formula I, X is a spacer moiety andPOLY is a water-soluble, non-peptidic oligomer.

Additional exemplary conjugates of the opioid agonists of Formula Iinclude those having the following structure:

wherein each of R¹, R², R³, R⁴, the dotted line (“---”), and Y¹ is aspreviously defined with respect to Formula I, X is a spacer moiety andPOLY is a water-soluble, non-peptidic oligomer.

Further additional exemplary conjugates of the opioid agonists ofFormula I include those having the following structure:

wherein each of R¹, R², R³, R⁴, Y¹ and R⁵ is as previously defined withrespect to Formula I, X is a spacer moiety and POLY is a water-soluble,non-peptidic oligomer.

Still further exemplary conjugates of the opioid agonists of Formula Iinclude those having the following structure:

wherein each of R¹, R², R³, R⁴, Y¹ and R⁵ is as previously defined withrespect to Formula I, X is a spacer moiety and POLY is a water-soluble,non-peptidic oligomer.

Additional exemplary conjugates of the opioid agonists of Formula Iinclude those having the following structure:

wherein each of R¹, R³, R⁴, the dotted line (“---”), Y¹ and R⁵ is aspreviously defined with respect to Formula I, X is a spacer moiety andPOLY is a water-soluble, non-peptidic oligomer.

Additional exemplary conjugates are encompassed by the followingformulae:

wherein, when present, each of R¹, R², R³, R⁴, the dotted line (“---”),Y¹ and R⁵ is as previously defined with respect to Formula I, and thevariable “n” is an integer from 1 to 30.

Exemplary conjugates of the opioid agonists of Formula II include thosehaving the following structure:

wherein:

N* is nitrogen;

Ar is selected from the group consisting of cyclohexyl, phenyl,halophenyl, methoxyphenyl, aminophenyl, pyridyl, furyl and thienyl;

Alk is selected from the group consisting of ethylene and propylene;

R_(II) is selected from the group consisting of lower alkyl, loweralkoxy, dimethylamino, cyclopropyl, 1-pyrrolidyl, morpholino (preferablylower alkyl such as ethyl);

R_(II)′ is selected from the group consisting of hydrogen, methyl andmethoxy;

R_(II)″ is selected from the group consisting of hydrogen and an organicradical (preferably lower alkyl);

X is a linker (e.g., a covalent bond “—” or one or more atoms); and

POLY is a water-soluble, non-peptidic oligomer.

With respect to Formula II-Ca, it will be understood that, depending onthe conditions, one or both of the amines—but more typically, the aminemarked with an asterisk (“N*”) in Formula II-Ca—can be protonated.

Additional exemplary conjugates of the opioid agonists of Formula IIinclude those having the following structure:

wherein:

Ar is selected from the group consisting of cyclohexyl, phenyl,halophenyl, methoxyphenyl, aminophenyl, pyridyl, furyl and thienyl;

Alk is selected from the group consisting of ethylene and propylene;

R_(II) is selected from the group consisting of lower alkyl, loweralkoxy, dimethylamino, cyclopropyl, 1-pyrrolidyl, morpholino (preferablylower alkyl such as ethyl);

R_(II)′ is selected from the group consisting of hydrogen, methyl andmethoxy;

R_(II)″ is selected from the group consisting of hydrogen and an organicradical (preferably lower alkyl);

X is a linker (e.g., a covalent bond “—” or one or more atoms); and

POLY is a water-soluble, non-peptidic oligomer.

With respect to Formula II-Cb, it will be understood that, depending onthe conditions, one or both of the amines—but more typically, the aminemarked with an asterisk (“N*”) in Formula II-Cb—can be protonated.

Additional exemplary conjugates of the opioid agonists of Formula IIinclude those having the following structure:

wherein:

Ar is selected from the group consisting of cyclohexyl, phenyl,halophenyl, methoxyphenyl, aminophenyl, pyridyl, furyl and thienyl;

Alk is selected from the group consisting of ethylene and propylene;

R_(II) is selected from the group consisting of lower alkyl, loweralkoxy, dimethylamino, cyclopropyl, 1-pyrrolidyl, morpholino (preferablylower alkyl such as ethyl);

R_(II)′ is selected from the group consisting of hydrogen, methyl andmethoxy;

R_(II)″ is selected from the group consisting of hydrogen and an organicradical (preferably lower alkyl);

each X is independently a linker (e.g., a covalent bond “—” or one ormore atoms); and

each POLY is independently a water-soluble, non-peptidic oligomer.

With respect to Formula II-Cc, it will be understood that, depending onthe conditions, one or both of the amines—but more typically, the aminemarked with an asterisk (“N*”) in Formula II-Cc—can be protonated.

Additional exemplary conjugates are encompassed by the followingformulae:

wherein the variable “n” is an integer from 1 to 30.

Additional conjugates include those provided below:

wherein, for each of the above conjugates, X is a linker (e.g., acovalent bond “—” or one or more atoms) and POLY is a water-soluble,non-peptidic oligomer.

An additional conjugate is provided below:

wherein:

R¹ is acyl

R² is selected from the group consisting of hydrogen, halogen,unsubstituted alkyl and alkyl substituted by halogen;

R³ is selected from the group consisting of halogen and alkoxy;

R⁵ is selected from the group consisting of hydroxyl, ester, alkoxy, andalkoxyalkyl;

A₁ is alkylene;

X is a linker; and

POLY is a water-soluble, non-peptidic oligomer.

The conjugates of the invention can exhibit a reduced blood-brainbarrier crossing rate. Moreover, the conjugates maintain at least about5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more of the bioactivity of theunmodified parent small molecule drug.

While it is believed that the full scope of the conjugates disclosedherein have been described, an optimally sized oligomer can bedetermined as follows.

First, an oligomer obtained from a monodisperse or bimodal water solubleoligomer is conjugated to the small molecule drug. Preferably, the drugis orally bioavailable, and on its own, exhibits a non-negligibleblood-brain barrier crossing rate. Next, the ability of the conjugate tocross the blood-brain barrier is determined using an appropriate modeland compared to that of the unmodified parent drug. If the results arefavorable, that is to say, if, for example, the rate of crossing issignificantly reduced, then the bioactivity of conjugate is furtherevaluated. Preferably, the compounds according to the invention maintaina significant degree of bioactivity relative to the parent drug, i.e.,greater than about 30% of the bioactivity of the parent drug, or evenmore preferably, greater than about 50% of the bioactivity of the parentdrug.

The above steps are repeated one or more times using oligomers of thesame monomer type but having a different number of subunits and theresults are compared.

For each conjugate whose ability to cross the blood-brain barrier isreduced in comparison to the non-conjugated small molecule drug, itsoral bioavailability is then assessed. Based upon these results, that isto say, based upon the comparison of conjugates of oligomers of varyingsize to a given small molecule at a given position or location withinthe small molecule, it is possible to determine the size of the oligomermost effective in providing a conjugate having an optimal balancebetween reduction in biological membrane crossing, oral bioavailability,and bioactivity. The small size of the oligomers makes such screeningsfeasible, and allows one to effectively tailor the properties of theresulting conjugate. By making small, incremental changes in oligomersize, and utilizing an experimental design approach, one can effectivelyidentify a conjugate having a favorable balance of reduction inbiological membrane crossing rate, bioactivity, and oralbioavailability. In some instances, attachment of an oligomer asdescribed herein is effective to actually increase oral bioavailabilityof the drug.

For example, one of ordinary skill in the art, using routineexperimentation, can determine a best suited molecular size and linkagefor improving oral bioavailability by first preparing a series ofoligomers with different weights and functional groups and thenobtaining the necessary clearance profiles by administering theconjugates to a patient and taking periodic blood and/or urine sampling.Once a series of clearance profiles have been obtained for each testedconjugate, a suitable conjugate can be identified.

Animal models (rodents and dogs) can also be used to study oral drugtransport. In addition, non-in vivo methods include rodent everted gutexcised tissue and Caco-2 cell monolayer tissue-culture models. Thesemodels are useful in predicting oral drug bioavailability.

To determine whether the opioid agonist or the conjugate of an opioidagonist and a water-soluble non-peptidic oligomer has activity as muopioid receptor agonist, it is possible to test such a compound. Forexample, K_(D) (binding affinity) and B_(max) (receptor number) can bedetermined using an approach modified from that described in Malatynskaet al. (1995) NeuroReport 6:613-616. Briefly, human mu receptors can berecombinantly expressed in Chinese hamster ovary cells. The radioligand[³H]-diprenorphine (30-50 Ci/mmol) with a final ligand concentration of[0.3 nM] can be used. Naloxone is used as a non-specific determinate[3.0 nM], a reference compound and positive control. Reactions arecarried out in 50 mM TRIS-HCl (pH 7.4) containing 5 mM MgCl₂, at 25° C.for 150 minutes. The reaction is terminated by rapid vacuum filtrationonto glass fiber filters. Radioactivity trapped onto filters isdetermined and compared to control values in order to ascertain anyinteractions of test compound with the cloned mu binding site.

Similar testing can be performed for kappa opioid receptor agonist. See,for example, Lahti et al. (1985) Eur. Jrnl. Pharmac. 109:281-284;Rothman et al. (1992) Peptides 13:977-987; Kinouchi et al. (1991) Eur.Jrnl. Pharmac. 207:135-141. Briefly, human kappa receptors can beobtained from guinea pig cerebellar membranes. The radioligand[³H]-U-69593 (40-60 Ci/mmol) with a final ligand concentration of [0.75nM] can be used. U-69593 is used as a non-specific determinate [1.0 μM],a reference compound and positive control. Reactions are carried out in50 mM HEPES (pH 7.4) at 30° C. for 120 minutes. The reaction isterminated by rapid vacuum filtration onto glass fiber filters.Radioactivity trapped onto filters is determined and compared to controlvalues in order to ascertain any interactions of test compound with thecloned kappa binding site.

The conjugates described herein include not only the conjugatesthemselves, but the conjugates in the form of a pharmaceuticallyacceptable salt as well. A conjugate as described herein can possess asufficiently acidic group, a sufficiently basic group, or bothfunctional groups, and, accordingly, react with any of a number ofinorganic bases, and inorganic and organic acids, to form a salt. Acidscommonly employed to form acid addition salts are inorganic acids suchas hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid,phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of such salts includethe sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Such bases useful in preparing the salts ofthis invention thus include sodium hydroxide, potassium hydroxide,ammonium hydroxide, potassium carbonate, and the like.

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Pharmaceutically acceptable acids or bases may be present as anexcipient in the preparation. Nonlimiting examples of acids that can beused include those acids selected from the group consisting ofhydrochloric acid, acetic acid, phosphoric acid, citric acid, malicacid, lactic acid, formic acid, trichloroacetic acid, nitric acid,perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, andcombinations thereof. Examples of suitable bases include, withoutlimitation, bases selected from the group consisting of sodiumhydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide,ammonium acetate, potassium acetate, sodium phosphate, potassiumphosphate, sodium citrate, sodium formate, sodium sulfate, potassiumsulfate, potassium fumerate, and combinations thereof.

The amount of the conjugate in the composition will vary depending on anumber of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container. Atherapeutically effective dose can be determined experimentally byrepeated administration of increasing amounts of the conjugate in orderto determine which amount produces a clinically desired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about5%-98% by weight, more preferably from about 15-95% by weight of theexcipient, with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsand general teachings regarding pharmaceutical compositions aredescribed in “Remington: The Science & Practice of Pharmacy”, 19^(th)ed., Williams & Williams, (1995), the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), and Kibbe, A. H.,Handbook of Pharmaceutical Excipients, 3^(rd) Edition, AmericanPharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical compositions can take any number of forms and theinvention is not limited in this regard. Exemplary preparations are mostpreferably in a form suitable for oral administration such as a tablet,caplet, capsule, gel cap, troche, dispersion, suspension, solution,elixir, syrup, lozenge, transdermal patch, spray, suppository, andpowder.

Oral dosage forms are preferred for those conjugates that are orallyactive, and include tablets, caplets, capsules, gel caps, suspensions,solutions, elixirs, and syrups, and can also comprise a plurality ofgranules, beads, powders or pellets that are optionally encapsulated.Such dosage forms are prepared using conventional methods known to thosein the field of pharmaceutical formulation and described in thepertinent texts.

Tablets and caplets, for example, can be manufactured using standardtablet processing procedures and equipment. Direct compression andgranulation techniques are preferred when preparing tablets or capletscontaining the conjugates described herein. In addition to theconjugate, the tablets and caplets will generally contain inactive,pharmaceutically acceptable carrier materials such as binders,lubricants, disintegrants, fillers, stabilizers, surfactants, coloringagents, and the like. Binders are used to impart cohesive qualities to atablet, and thus ensure that the tablet remains intact. Suitable bindermaterials include, but are not limited to, starch (including corn starchand pregelatinized starch), gelatin, sugars (including sucrose, glucose,dextrose and lactose), polyethylene glycol, waxes, and natural andsynthetic gums, e.g., acacia sodium alginate, polyvinylpyrrolidone,cellulosic polymers (including hydroxypropyl cellulose, hydroxypropylmethylcellulose, methyl cellulose, microcrystalline cellulose, ethylcellulose, hydroxyethyl cellulose, and the like), and Veegum. Lubricantsare used to facilitate tablet manufacture, promoting powder flow andpreventing particle capping (i.e., particle breakage) when pressure isrelieved. Useful lubricants are magnesium stearate, calcium stearate,and stearic acid. Disintegrants are used to facilitate disintegration ofthe tablet, and are generally starches, clays, celluloses, algins, gums,or crosslinked polymers. Fillers include, for example, materials such assilicon dioxide, titanium dioxide, alumina, talc, kaolin, powderedcellulose, and microcrystalline cellulose, as well as soluble materialssuch as mannitol, urea, sucrose, lactose, dextrose, sodium chloride, andsorbitol. Stabilizers, as well known in the art, are used to inhibit orretard drug decomposition reactions that include, by way of example,oxidative reactions.

Capsules are also preferred oral dosage forms, in which case theconjugate-containing composition can be encapsulated in the form of aliquid or gel (e.g., in the case of a gel cap) or solid (includingparticulates such as granules, beads, powders or pellets). Suitablecapsules include hard and soft capsules, and are generally made ofgelatin, starch, or a cellulosic material. Two-piece hard gelatincapsules are preferably sealed, such as with gelatin bands or the like.

Included are parenteral formulations in the substantially dry form(typically as a lyophilizate or precipitate, which can be in the form ofa powder or cake), as well as formulations prepared for injection, whichare typically liquid and requires the step of reconstituting the dryform of parenteral formulation. Examples of suitable diluents forreconstituting solid compositions prior to injection includebacteriostatic water for injection, dextrose 5% in water,phosphate-buffered saline, Ringer's solution, saline, sterile water,deionized water, and combinations thereof.

In some cases, compositions intended for parenteral administration cantake the form of nonaqueous solutions, suspensions, or emulsions, eachtypically being sterile. Examples of nonaqueous solvents or vehicles arepropylene glycol, polyethylene glycol, vegetable oils, such as olive oiland corn oil, gelatin, and injectable organic esters such as ethyloleate.

The parenteral formulations described herein can also contain adjuvantssuch as preserving, wetting, emulsifying, and dispersing agents. Theformulations are rendered sterile by incorporation of a sterilizingagent, filtration through a bacteria-retaining filter, irradiation, orheat.

The conjugate can also be administered through the skin usingconventional transdermal patch or other transdermal delivery system,wherein the conjugate is contained within a laminated structure thatserves as a drug delivery device to be affixed to the skin. In such astructure, the conjugate is contained in a layer, or “reservoir,”underlying an upper backing layer. The laminated structure can contain asingle reservoir, or it can contain multiple reservoirs.

The conjugate can also be formulated into a suppository for rectaladministration. With respect to suppositories, the conjugate is mixedwith a suppository base material which is (e.g., an excipient thatremains solid at room temperature but softens, melts or dissolves atbody temperature) such as coca butter (theobroma oil), polyethyleneglycols, glycerinated gelatin, fatty acids, and combinations thereof.Suppositories can be prepared by, for example, performing the followingsteps (not necessarily in the order presented): melting the suppositorybase material to form a melt; incorporating the conjugate (either beforeor after melting of the suppository base material); pouring the meltinto a mold; cooling the melt (e.g., placing the melt-containing mold ina room temperature environment) to thereby form suppositories; andremoving the suppositories from the mold.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with the conjugate. The method comprisesadministering, generally orally, a therapeutically effective amount ofthe conjugate (preferably provided as part of a pharmaceuticalpreparation). Other modes of administration are also contemplated, suchas pulmonary, nasal, buccal, rectal, sublingual, transdermal, andparenteral. As used herein, the term “parenteral” includes subcutaneous,intravenous, intra-arterial, intraperitoneal, intracardiac, intrathecal,and intramuscular injection, as well as infusion injections.

In instances where parenteral administration is utilized, it may benecessary to employ somewhat bigger oligomers than those describedpreviously, with molecular weights ranging from about 500 to 30K Daltons(e.g., having molecular weights of about 500, 1000, 2000, 2500, 3000,5000, 7500, 10000, 15000, 20000, 25000, 30000 or even more).

The method of administering may be used to treat any condition that canbe remedied or prevented by administration of the particular conjugate.Those of ordinary skill in the art appreciate which conditions aspecific conjugate can effectively treat. The actual dose to beadministered will vary depend upon the age, weight, and generalcondition of the subject as well as the severity of the condition beingtreated, the judgment of the health care professional, and conjugatebeing administered. Therapeutically effective amounts are known to thoseskilled in the art and/or are described in the pertinent reference textsand literature. Generally, a therapeutically effective amount will rangefrom about 0.001 mg to 1000 mg, preferably in doses from 0.01 mg/day to750 mg/day, and more preferably in doses from 0.10 mg/day to 500 mg/day.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

One advantage of administering the conjugates of the present inventionis that a reduction in first pass metabolism may be achieved relative tothe parent drug. Such a result is advantageous for many orallyadministered drugs that are substantially metabolized by passage throughthe gut. In this way, clearance of the conjugate can be modulated byselecting the oligomer molecular size, linkage, and position of covalentattachment providing the desired clearance properties. One of ordinaryskill in the art can determine the ideal molecular size of the oligomerbased upon the teachings herein. Preferred reductions in first passmetabolism for a conjugate as compared to the correspondingnonconjugated small drug molecule include: at least about 10%, at leastabout 20%, at least about 30; at least about 40; at least about 50%; atleast about 60%, at least about 70%, at least about 80% and at leastabout 90%.

Thus, the invention provides a method for reducing the metabolism of anactive agent. The method comprises the steps of: providing monodisperseor bimodal conjugates, each conjugate comprised of a moiety derived froma small molecule drug covalently attached by a stable linkage to awater-soluble oligomer, wherein said conjugate exhibits a reduced rateof metabolism as compared to the rate of metabolism of the smallmolecule drug not attached to the water-soluble oligomer; andadministering the conjugate to a patient. Typically, administration iscarried out via one type of administration selected from the groupconsisting of oral administration, transdermal administration, buccaladministration, transmucosal administration, vaginal administration,rectal administration, parenteral administration, and pulmonaryadministration.

Although useful in reducing many types of metabolism (including bothPhase I and Phase II metabolism) can be reduced, the conjugates areparticularly useful when the small molecule drug is metabolized by ahepatic enzyme (e.g., one or more of the cytochrome P450 isoforms)and/or by one or more intestinal enzymes.

All articles, books, patents, patent publications and other publicationsreferenced herein are incorporated by reference in their entireties. Inthe event of an inconsistency between the teachings of thisspecification and the art incorporated by reference, the meaning of theteachings in this specification shall prevail.

EXPERIMENTAL

It is to be understood that while the invention has been described inconjunction with certain preferred and specific embodiments, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All chemical reagents referred to in the appended examples arecommercially available unless otherwise indicated. The preparation ofPEG-mers is described in, for example, U.S. Patent ApplicationPublication No. 2005/0136031.

All ¹H NMR (nuclear magnetic resonance) data was generated by a NMRspectrometer manufactured by Bruker (MHz≧200). A list of certaincompounds as well as the source of the compounds is provided below.

Example 1 Preparation of an Oligomer-Nalbuphine Conjugates—“Approach A”

PEG-Nalbuphine was prepared using a first approach. Schematically, theapproach followed for this example is shown below.

Desalting of Nalbuphine Hydrochloride Dihydrate:

Nalbuphine hydrochloride dihydrate (600 mg, from Sigma) was dissolved inwater (100 mL). Saturated aqueous K₂CO₃ was added and then adjusted thepH to 9.3 with 1 N HCl solution, saturated with sodium chloride. Thesolution was extracted with dichloromethane (5×25 mL). The combinedorganic solution was washed with brine (100 mL), dried over Na₂SO₄,concentrated to dryness and dried under high vacuum to yield nalbuphine(483.4 mg, 97% recovery). The product was confirmed by ¹H-NMR in CDCl₃.

Synthesis of 3-O-mPEG₃-Nalbuphine (2) (n=3)

Nalbuphine (28.5 mg, 0.08 mmol) was dissolved in a mixture of acetone (2mL) and toluene (1.5 mL). Potassium carbonate (21 mg, 0.15 mmol) wasadded, followed by an addition of mPEG₃-Br (44.5 mg, 0.20 mmol) at roomtemperature. The resulting mixture was stirred at room temperature for27.5 hours. More potassium carbonate (24 mg, 0.17 mmol) was added. Themixture was heated with CEM microwave such that 60° C. for 20 minuteswas achieved, and then such that 100° C. for 30 minutes was achieved.DMF (0.2 mL) was added. The mixture was heated with microwave at 60° C.for 20 minutes, at 100° C. for 30 minutes. The reaction was concentratedto remove the organic solvents, the residue was mixed with water (10mL), extracted with dichloromethane (4×15 mL). The combined organicsolution was washed with brine, dried over Na₂SO₄, concentrated. Thecrude product was checked with HPLC and LC-MS. The residue was mixedagain with water (10 mL), adjusted the pH to 2.3 with 1N HCl, washedwith dichloromethane (2×15 mL). The aqueous solution was adjusted to pH10.4 with 0.2 N NaOH, extracted with dichloromethane (4×15 mL). Thecombined organic solution was washed with brine, dried over Na₂SO₄,concentrated. The residue was purified by Biotage flash columnchromatography with 0-10% MeOH in dichloromethane resulting in thedesired product 3-O-mPEG₃-nalbuphine (2) (n=3) (32.7 mg) in 81% yield.The product was confirmed by ¹H-NMR, LC-MS.

Synthesis of 3-O-mPEG₄-Nalbuphine (2) (n=4)

A mixture of nalbuphine (96 mg, 0.27 mmol) and mPEG₄-OMs (131 mg, 0.46mmol) in acetone (8 mL) in the presence of potassium carbonate (113 mg,0.82 mmol) was heated to reflux for 16 hours, cooled to roomtemperature, filtered and the solid was washed with acetone and DCM. Thesolution was collected and concentrated to dryness. The residue waspurified by Biotage automatic flash column chromatography with 0-10%MeOH in dichloromethane to result in the product 3-O-mPEG₄-nalbuphine 2(n=4) (109 mg) in 74% yield. The product was confirmed by ¹H-NMR, LC-MS.

Synthesis of 3-O-mPEG₅-Nalbuphine (2) (n=5)

A mixture of nalbuphine (78.3 mg, 0.22 mmol) and mPEG₅-OMs (118 mg, 0.36mmol) in acetone (8 mL) in the presence of potassium carbonate (93 mg,0.67 mmol) was heated to reflux for 16 hours, cooled to roomtemperature, filtered and the solid was washed with acetone and DCM. Thesolution was collected and concentrated to dryness. The residue waspurified by Biotage automatic flash column chromatography with 0-10%MeOH in dichloromethane to result in the product 3-O-mPEG₅-nalbuphine(2) (n=5) (101 mg) in 76% yield. The product was confirmed by ¹H-NMR,LC-MS.

Synthesis of 3-O-mPEG₆-Nalbuphine (2) (n=6)

A mixture of nalbuphine (89.6 mg, 0.25 mmol) and mPEG₆-OMs (164 mg, 0.44mmol) in acetone (8 mL) in the presence of potassium carbonate (98 mg,0.71 mmol) was heated to reflux for 18 hours, cooled to roomtemperature, filtered and the solid washed with acetone and DCM. Thesolution was collected and concentrated to dryness. The residue waspurified by Biotage automatic flash column chromatography with 0-10%MeOH in dichloromethane to result in the product 3-O-mPEG₆-nalbuphine(2) (n=6) (144 mg) in 91% yield. The product was confirmed by ¹H-NMR,LC-MS.

Synthesis of 3-O-mPEG₇-Nalbuphine (2) (n=7)

A mixture of nalbuphine (67 mg, 0.19 mmol) and mPEG₇-Br (131 mg, 0.33mmol) in acetone (10 mL) in presence of potassium carbonate (67 mg, 0.49mmol) was heated to reflux for 6 hours, cooled to room temperature,filtered and the solid washed and dichloromethane. The solution wasconcentrated to dryness. The residue was purified by Biotage automaticflash column chromatography with 2-10% MeOH in dichloromethane to resultin the product 3-O-mPEG₇-nalbuphine (2) (n=7) (40.6 mg). The product wasconfirmed by ¹H-NMR, LC-MS.

Synthesis of 3-O-mPEG₈-Nalbuphine (2) (n=8)

A mixture of nalbuphine (60 mg, 0.17 mmol) and mPEG₈-Br (105.7 mg, 0.24mmol) in the presence of potassium carbonate (40.8 mg, 0.30 mmol) intoluene/DMF (3 mL/0.3 mL) was heated with CEM microwave such that 100°C. for 30 minutes was achieved. Then acetone (1 mL) was added. After themixture was heated with CEM microwave such that 100° C. for 90 minuteswas achieved, more of K₂CO₃ (31 mg, 0.22 mmol) and mPEG₈-Br (100 mg,0.22 mmol) were added. The mixture was heated with CEM microwave suchthat 100° C. for 60 minutes was achieved. mPEG₈-Br (95 mg, 0.21 mmol)was added again. The mixture was heated again with CEM microwave suchthat 100° C. for 30 minutes was achieved. The reaction mixture wasconcentrated under reduce pressure. The residue was mixed with water (2mL) and brine (10 mL). The pH of the solution was adjusted to 1.56 with1 N HCl, extracted with dichloromethane (3×20 mL). The combined organicsolution was dried with Na₂SO₄, concentrated to yield residue I (amixture of the desired product and precursor material). The aqueoussolution was changed to pH 10.13 with 0.2 N NaOH, extracted withdichloromethane (4×15 mL). The organic solution was washed with brine,dried over Na₂SO₄, concentrated to result in residue II (19.4 mg), whichcontained the product and the starting material nalbuphine. The residueI was purified by Biotage automatic flash column chromatography with2-10% MeOH in dichloromethane to result in the product3-O-mPEG₈-nalbuphine (2) (n=8) (44.6 mg). The product was confirmed by¹H-NMR. LC-MS.

Example 2 Preparation of an Oligomer-Nalbuphine Conjugates—“Approach B”

PEG-Nalbuphine was prepared using a second approach. Schematically, theapproach followed for this example is shown below.

Synthesis of 3-O-MEM-Nalbuphine (3)

Nalbuphine (321.9 mg, 0.9 mmol) was dissolved in acetone/toluene (19mL/8 mL). Then potassium carbonate (338 mg, 2.45 mmol) was added,followed by an addition of MEMCl (160 μL, 1.41 mmol). The resultingmixture was stirred at room temperature for 21 hours MeOH (0.3 mL) wasadded to quench the reaction. The reaction mixture was concentratedunder reduced pressure to dryness. The residue was mixed with water (5mL) and brine (15 mL), extracted with dichloromethane (3×15 mL). Thecombined organic solution was washed with brine, dried over Na₂SO₄,concentrated. The residue was separated by Biotage automatic flashcolumn chromatography with 2-10% MeOH in dichloromethane to result inthe product 3-O-MEM-nalbuphine (3) (341 mg) and the starting materialnalbuphine (19.3 mg). The product was confirmed by ¹H-NMR, LC-MS.

Synthesis of 6-O-mPEG₃-3-O-MEM-Nalbuphine (4) (n=3)

A 20-mL vial was placed with 3-O-MEM-nalbuphine (3) (85 mg, 0.19 mmol)and toluene (15 mL). The mixture was concentrated to remove 7 mL oftoluene. Anhydrous DMF (0.2 mL) was added. The vial was flashed withnitrogen. NaH (60% dispersion in mineral oil, 21 mg, 0.53 mmol) wasadded, followed by an addition of mPEG₃-OMs (94 mg. 0.39 mmol). Afterthe resulting mixture was heated at 45° C. for 22.5 hours, more of NaH(22 mg, 0.55 mmol) was added. The mixture was heated at 45° C. foranother six hours, NaH (24 mg) was added and the mixture was heated at45° C. for another 19 hours. When the mixture was cooled to roomtemperature, saturated NaCl aqueous solution (1 mL) was added to quenchthe reaction. The mixture was diluted with water (10 mL), extracted withEtOAc (4×15 mL). The combined organic solution was washed with brine,dried over Na₂SO₄, concentrated. The residue was separated by Biotageautomatic flash column chromatography with 0-10% MeOH in dichloromethaneto result in the product 6-O-mPEG₃-3-O-MEM-nalbuphine (4) (n=3) (79.4mg) in 71% yield. The product was confirmed by ¹H-NMR, LC-MS.

Synthesis of 6-O-mPEG₃-Nalbuphine (5) (n=3)

6-O-mPEG₃-3-O-MEM-nalbuphine (4) (79.4 mg) was stirred in 2 M HCl inmethanol at room temperature for six hours. The mixture was diluted withwater (5 mL), and concentrated to removed the methanol. The aqueoussolution was washed with dichloromethane (5 mL), and the pH of thesolution was adjusted to 9.35 with 0.2 N NaOH and solid NaHCO₃,extracted with dichloromethane (4×30 mL). The combined organic solutionwas washed with brine, dried over Na₂SO₄, concentrated to result in theproduct 6-O-mPEG₃-nalbuphine (5) (n=3) (62.5 mg) in 93% yield. Theproduct was confirmed by ¹H-NMR, LC-MS.

Synthesis of 6-O-mPEG₄-3-O-MEM-Nalbuphine (4) (n=4)

A 50-mL round-flask was placed with 3-O-MEM-nalbuphine (3) (133.8 mg,0.3 mmol) and mPEG₄-OMs (145 mg, 0.51 mmol) and toluene (20 mL). Themixture was concentrated to remove about 12 mL of toluene. Anhydrous DMF(0.2 mL) was added. NaH (60% dispersion in mineral oil, 61 mg, 1.52mmol) was added. After the resulting mixture was heated at 45° C. for21.5 hours, more of NaH (30 mg, 0.75 mmol) was added. The mixture washeated at 45° C. for another five hours. When the mixture was cooled toroom temperature, saturated NaCl aqueous solution (1 mL) was added toquench the reaction. The mixture was diluted with water (15 mL), andextracted with EtOAc (4×15 mL). The combined organic solution was washedwith brine, dried over Na₂SO₄, concentrated. The residue was separatedby Biotage automatic flash column chromatography on silica gel with0-10% MeOH in dichloromethane to result in the product6-O-mPEG₄-3-O-MEM-nalbuphine (4) (n=4) (214.4 mg). The ¹H-NMR showedsome mPEG₄-OMs in the product. No attempt was made for furtherpurification. The product was confirmed by ¹H-NMR, LC-MS.

Synthesis of 6-O-mPEG₄-Nalbuphine (5) (n=4)

6-O-mPEG₄-3-O-MEM-nalbuphine (4) (214.4 mg) was stirred in 2 M HCl inmethanol (30 mL) at room temperature for 6 hours. The mixture wasdiluted with water (5 mL), and concentrated to removed the methanol. Theaqueous solution was adjusted to 9.17 with 1 N NaOH, extracted withdichloromethane (4×25 mL). The combined organic solution was washed withbrine, dried over Na₂SO₄, and concentrated. The residue was purified byflash column chromatography on silica gel using 3-8% MeOH/DCM (Biotage)to result in the pure product 6-O-mPEG₄-nalbuphine (5) (n=4) (90.7 mg),along with some impure product. The product was confirmed by ¹H-NMR.LC-MS. The impure part was dissolved in DCM (˜1.5 mL). 1 N HCl in ether(20 mL) was added, centrifuged. The residue was collected andredissolved in DCM (25 mL). The DCM solution was washed with aq. 5%NaHCO₃ (20 mL), brine (2×30 mL), dried over Na₂SO₄, concentrated toafford another part pure product (24.8 mg).

Synthesis of 6-O-mPEG₅-3-O-MEM-Nalbuphine (4) (n=5)

A 50-mL round-flask was placed with 3-O-MEM-nalbuphine (3) (103.9 mg,0.23 mmol), mPEG₅-OMs (151 mg, 0.46 mmol) and toluene (38 mL). Themixture was concentrated to remove about 20 mL of toluene. Anhydrous DMF(0.5 mL) was added. NaH (60% dispersion in mineral oil, 102 mg, 2.55mmol) was added. After the resulting mixture was heated at 45° C. for 18hours, more of NaH (105 mg) was added. The mixture was heated at 45° C.for another 5.5 hours. NaH (87 mg) was added and the mixture was heatedat 45° C. for another 17.5 hours. When the mixture was cooled to roomtemperature, saturated NaCl aqueous solution (3 mL) was added to quenchthe reaction. The mixture was diluted with water (10 mL), extracted withEtOAc (4×20 mL). The combined organic solution was washed with brine,dried over Na₂SO₄, concentrated. The residue was separated by Biotageautomatic flash column chromatography on silica gel with 3-8% MeOH indichloromethane to result in the product 6-O-mPEG₅-3-O-MEM-nalbuphine(4) (n=5).

Synthesis of 6-O-mPEG₅-Nalbuphine (5) (n=5)

The above 6-O-mPEG₅-3-O-MEM-nalbuphine (4) was stirred in 2 M HCl inmethanol (30 mL) at room temperature for 2.5 hour. The mixture wasdiluted with water (5 mL), concentrated to removed the methanol. Theaqueous solution was adjusted to 9.19 with 1 N NaOH, extracted withdichloromethane (4×15 mL). The combined organic solution was washed withbrine, dried over Na₂SO₄, concentrated. After purification with flashcolumn chromatography on silica, mPEG₅-OMs was observed in ¹H-NMR. Theresidue was dissolved in DCM (˜1 mL). 1 N HCl in ether (18 mL) wasadded, centrifuged. The residue was collected and redissolved in DCM (25mL). The DCM solution was washed with aq. 5% NaHCO₃ (2×20 mL), brine(2×30 mL), dried over Na₂SO₄, concentrated. The residue was separated byBiotage automatically flash column chromatography on silica gel with4-8% MeOH in dichloromethane to result in the product6-O-mPEG₅-nalbuphine (5) (n=5) (55 mg).

Synthesis of 6-O-mPEG₆-3-O-MEM-Nalbuphine (4) (n=6)

3-O-MEM-nalbuphine (3) (77.6 mg, 0.17 mmol) and mPEG₆-OMs (199 mg, 0.53mmol) was dissolved in toluene (20 mL). The mixture was concentrated toremove about 12 mL of toluene. Anhydrous DMF (0.2 mL) was added,followed by an addition of NaH (60% dispersion in mineral oil, 41 mg,1.03 mmol). After the resulting mixture was heated at 45° C. for 23hours, more of NaH (46 mg) was added. The mixture was heated at 45° C.for another 24 hours. When the mixture was cooled to room temperature,saturated NaCl aqueous solution (5 mL) was added to quench the reaction.The mixture was diluted with water (10 mL), extracted with EtOAc (4×15mL). The combined organic solution was washed with brine, dried overNa₂SO₄, concentrated. The residue was directly used for the next step.

Synthesis of 6-O-mPEG₆-Nalbuphine (5) (n=6)

The above 6-O-mPEG₆-3-O-MEM-nalbuphine (4) was stirred in 2 M HCl inmethanol (30 mL) at room temperature for 20 hours. The mixture wasdiluted with water (5 mL), concentrated to removed the methanol. Theaqueous solution was adjusted to 9.30 with 1 N NaOH, extracted withdichloromethane (5×20 mL). The combined organic solution was washed withbrine, dried over Na₂SO₄, concentrated. The residue was dissolved in DCM(˜1 mL). 1 N HCl in ether (20 mL) was added, centrifuged. The residuewas collected and redissolved in DCM (40 mL). The DCM solution waswashed with aq. 5% NaHCO₃ (2×20 mL), water (30 mL), brine (2×30 mL),dried over Na₂SO₄, concentrated to result in the product6-O-mPEG₆-nalbuphine (5) (n=6) (68 mg).

Synthesis of 6-O-mPEG₇-3-O-MEM-Nalbuphine (4, n=7)

A 50-mL round-flask was placed with 3-O-MEM-nalbuphine (3) (82.8 mg,0.186 mmol), mPEG₇-Br (151 mg, 0.46 mmol) and toluene (15 mL). Themixture was concentrated to remove about 9 mL of toluene. Anhydrous DMF(0.2 mL) was added. NaH (60% dispersion in mineral oil, 50 mg, 1.25mmol) was added. After the resulting mixture was heated at 45° C. for22.5 hours, more of NaH (38 mg, 0.94 mmol) was added. The mixture washeated at 45° C. for another 5 hours. When the mixture was cooled toroom temperature, saturated NaCl aqueous solution (5 mL) was added toquench the reaction. The mixture was diluted with water (10 mL), andextracted with EtOAc (4×10 mL). The combined organic solution was washedwith brine, dried over Na₂SO₄, concentrated. The residue was directlyused for the next step.

Synthesis of 6-O-mPEG₇-Nalbuphine (5) (n=7)

The above 6-O-mPEG₇-3-O-MEM-nalbuphine (4) was stirred in 2 M HCl inmethanol (20 mL) at room temperature for 20 hours. The mixture wasdiluted with water, and concentrated to remove the methanol. The aqueoussolution was adjusted to 9.30 with NaHCO₃ and 0.2 N NaOH, extracted withdichloromethane (4×20 mL). The combined organic solution was washed withbrine, dried over Na₂SO₄, concentrated. The residue was purified withflash column chromatography on silica gel and washed with DCM at acidiccondition, adjusted the pH to 9.35, extracted with DCM. The product wasstill contaminated with small PEG. The residue was dissolved in DCM (˜2mL). 1 N HCl in ether (10 mL) was added, centrifuged. The residue wascollected and redissolved in DCM (10 mL). The DCM solution was washedwith aq. 5% NaHCO₃, brine, dried over Na₂SO₄, concentrated to result inthe product 6-O-mPEG₇-nalbuphine (5) (n=7) (49 mg).

Synthesis of 6-O-mPEG₈-3-O-MEM-Nalbuphine (4) (n=8)

A 50-mL round-flask was placed with 3-O-MEM-nalbuphine (3) (80.5 mg.0.181 mmol), mPEG₈-Br (250 mg, 0.56 mmol) and toluene (15 mL). Themixture was concentrated to remove about 6 mL of toluene. Anhydrous DMF(0.2 mL) was added. NaH (60% dispersion in mineral oil, 49 mg, 1.23mmol) was added. The resulting mixture was heated at 45° C. for 23hours, the mixture was cooled to room temperature, saturated NaClaqueous solution (5 mL) and water (10 mL) was added to quench thereaction. The mixture was extracted with EtOAc (4×20 mL). The combinedorganic solution was washed with brine, dried over Na₂SO₄, concentrated.The residue was directly used for the next step.

Synthesis of 6-O-mPEG₈-Nalbuphine (5) (n=8)

The above 6-O-mPEG₈-3-O-MEM-nalbuphine (4) was stirred in 2 M HCl inmethanol (20 mL) at room temperature for 17 hours. The mixture wasdiluted with water, concentrated to remove the methanol. The aqueoussolution was adjusted to 9.32 with NaHCO₃ and 0.2 N NaOH, extracted withdichloromethane (4×20 mL). The combined organic solution was washed withbrine, dried over Na₂SO₄, concentrated. The residue was dissolved in DCM(˜1 mL). 1 N HCl in ether (20 mL) was added, centrifuged. The residuewas collected and redissolved in DCM (30 mL). The DCM solution waswashed with aq. 5% NaHCO₃ (60 mL), water (30 mL), brine (30 mL), driedover Na₂SO₄, concentrated. The residue was purified with flash columnchromatography on silica gel using 0-10% methanol in dichloromethane toresult in the product 6-O-mPEG₈-nalbuphine (5) (n=8) (78.4 mg).

Synthesis of 6-O-mPEG₉-3-O-MEM-Nalbuphine (4) (n=9)

A 50-mL round-flask was placed with 3-O-MEM-nalbuphine (3) (120 mg, 0.27mmol), mPEG₉-OMs (245 mg, 0.48 mmol) and toluene (20 mL). The mixturewas concentrated to remove about 10 mL of toluene. NaH (60% dispersionin mineral oil, 63 mg, 1.57 mmol) was added, followed by an addition ofanhydrous DMF (0.5 mL). The resulting mixture was heated at 45 OC for 17hours. More of NaH (60% dispersion in mineral oil, 60 mg) was addedbased on the HPLC results, and then the mixture was heated at 45 OC foranother 5.5 hours. The mixture was cooled to room temperature, saturatedNaCl aqueous solution (2 mL) and water (15 mL) was added to quench thereaction. The mixture was extracted with EtOAc (4×20 mL). The combinedorganic solution was washed with brine, dried over Na₂SO₄, concentrated.The residue was purified by flash column chromatography on silica gelusing 3-8% methanol in dichloromethane (biotage) to afford the product6-O-mPEG₉-3-MEM-O-nalbuphine (207 mg) in 90% yield.

Synthesis of 6-O-mPEG₉-Nalbuphine (5) (n=9)

The above 6-O-mPEG₉-3-O-MEM-nalbuphine (4) (207 mg, 0.24 mmol) wasstirred in 2 M HCl in methanol (33 mL) at room temperature for 17 hours.The mixture was diluted with water, and concentrated to remove themethanol. The aqueous solution was adjusted to 9.16 with 1 N NaOH, andextracted with dichloromethane (4×25 mL). The combined organic solutionwas washed with brine, dried over Na₂SO₄, concentrated. The residue waspurified with flash column chromatography on silica gel using 3-8%methanol in dichloromethane to result in the product6-O-mPEG₉-nalbuphine (4) (n=9) (129.3 mg) in 70% yield.

Example 3 Preparation of an Oligomer-Nalbuphine Conjugates—“Approach C”

PEG-Nalbuphine was prepared using a third approach. Schematically, theapproach followed for this example is shown below.

Synthesis of TrO-PEG₅-OH (7) (n=5)

PEG₅-di-OH (6) (n=5) (5.88 g, 24.19 mmol) was dissolved in toluene (30mL), and concentrated to remove toluene under reduced pressure. Theresidue was dried under high vacuum. Anhydrous DMF (40 mL) was added,followed by an addition of DMAP (0.91 g, 7.29 mmol) and TrCl (tritylchloride) (1.66 g, 5.84 mmol). The resulting mixture was heated at 50°C. for 22 hours. The reaction was concentrated to remove the solvents(high vacuum, 50° C.). The residue was mixed with water, and extractedwith EtOAc (3×25 mL). The combined organic solution was washed withbrine, dried over Na₂CO₃, concentrated. The residue was purified withflash column chromatography on silica gel to result in 1.29 g of productin 46% yield. The product was confirmed with ¹H-NMR in CDCl₃.

Synthesis of TrO-PEG_(n)-OH (7) (n=various)

Following a similar procedure for the preparation of TrO-PEG₅-OH, otherTrO-PEG_(n)-OH were synthesized from the corresponding PEG_(n)-di-OH.

Synthesis of TrO-PEG₅-OMs (8) (n=5)

Methanesulfonyl chloride (0.35 mL, 4.48 mmol) was added dropwise to astirred solution of TrO-PEG₅-OH (8) (n=5) (1.29 g, 2.68 mmol) andtriethylamine (0.9 mL, 6.46 mmol) in dichloromethane (15 mL) at 0° C.After the addition, the resulting solution was stirred at roomtemperature for 16.5 hours. Water was added to quench the reaction. Theorganic phase was separated and the aqueous solution was extracted withdichloromethane (10 mL). The combined organic solution was washed withbrine (3×30 mL), dried over Na₂SO₄ and concentrated to afford theproduct as oil (1.16 g) in 78% yield. The product (8) (n=5) wasconfirmed with ¹H-NMR in CDCl₃.

Synthesis of TrO-PEG_(n)-OMs (8) (n=various)

Following a similar procedure for the preparation of TrO-PEG₅-OMs, otherTrO-PEG_(n)-OMs were synthesized from the corresponding TrO-PEG_(n)-OH.

Synthesis of 3-O-MEM-6-O-TrO-PEG₄-nalbuphine (9) (n=4)

A round-flask was placed with 3-O-MEM-nalbuphine (3) (120 mg, 0.27 mmol)[previously prepared in accordance with the synthesis of compound (3)provided in Example 2], TrO-PEG₄-OMs (8) (n=4) (143.4 mg, 0.28 mmol) andtoluene (40 mL). The mixture was concentrated to remove about 30 mL oftoluene. NaH (60% dispersion in mineral oil, 150 mg, 3.75 mmol) wasadded, followed by an addition of anhydrous DMF (0.2 mL). The resultingmixture was heated at 45° C. for 4.5 hours. More of NaH (60% dispersionin mineral oil, 146 mg) was added, and the mixture was stirred at 45 OCfor another 18 hours. The mixture was cooled to room temperature, wassaturated with NaCl aqueous solution (2 mL), and water (15 mL) was addedto quench the reaction. The mixture was extracted with EtOAc (4×20 mL).The combined organic solution was washed with brine, dried over Na₂SO₄,and concentrated. The residue was purified by flash columnchromatography on silica gel using 0-10% methanol in dichloromethane(Biotage) to afford the product 3-O-MEM-6-O-TrO-PEG₄-nalbuphine (9)(n=4) (˜150 mg).

Synthesis of 6-O—HO-PEG₄-Nalbuphine (10) (n=4)

The above 6-O-TrO-PEG₄-3-O-MEM-nalbuphine (9) (n=4) (150 mg) was stirredin 2 M HCl in methanol (12 mL) at room temperature for one day. Themixture was diluted with water, and concentrated to remove the methanol.The aqueous solution was adjusted to PH 9.08 with NaOH, and extractedwith EtOAc (3×20 mL). The combined organic solution was washed withbrine, dried over Na₂SO₄, and concentrated. The residue was purifiedwith flash column chromatography on silica gel to result in the product6-O—OH-PEG₄-nalbuphine (10) (n=4) (26.9 mg). The product was analyzedwith ¹H-NMR, LC-Ms, HPLC.

Synthesis of 3-O-MEM-6-O-TrO-PEG₅-nalbuphine (9) (n=5)

A round-flask was placed with 3-O-MEM-nalbuphine (3) (318 mg, 0.71 mmol)[previously prepared in accordance with the synthesis of compound (3)provided in Example 2], TrO-PEG₅-OMs (8) (n=5) (518.5 mg, 0.93 mmol) andtoluene (100 mL). The mixture was concentrated to remove about 75 mL oftoluene. NaH (60% dispersion in mineral oil, 313 mg, 7.8 mmol) wasadded, followed by an addition of anhydrous DMF (1.0 mL). The resultingmixture was stirred at room temperature for 30 minutes, and then at 60OC for 19.5 hours. The mixture was cooled to room temperature, wassaturated with NaCl aqueous solution (5 mL), and water (5 mL) was addedto quench the reaction. The organic phase was separated and the aqueouswas extracted with EtOAc. The combined organic solution was washed withbrine, dried over Na₂SO₄, concentrated. The residue was purified byflash column chromatography on silica gel using 0-10% methanol indichloromethane (Biotage) to afford the product3-O-MEM-6-O-TrO-PEG₅-nalbuphine (718 mg). The product (9) (n=5) wasimpure, was used for the next step without further purification.

Synthesis of 6-O—HO-PEG₅-Nalbuphine (10) (n=5)

The above 6-O-TrO-PEG₅-3-O-MEM-nalbuphine (9) (n=5) (718 mg) was stirredin 2 M HCl in methanol (30 mL) at room temperature for 19 hours. Themixture was diluted with water, and concentrated to remove the methanol.The aqueous solution was adjusted to PH 9.16 with NaOH, extracted withDCM (3×20 mL). The combined organic solution was washed with brine,dried over Na₂SO₄, and concentrated. The residue was purified twice withflash column chromatography on silica gel to afford very pure product6-O—HO-PEG₅-nalbuphine 10 (n=5) (139 mg) and less pure product (48 mg).The product was analyzed with ¹H-NMR. LC-Ms, HPLC.

Example 4 Receptor Binding of PEG-Nalbuphine Conjugates

Using conventional receptor binding assay techniques, several moleculeswere assayed to determine binding activity at kappa, mu and delta opioidsubtypes of opioid receptors.

Briefly, the receptor binding affinity of the nalbuphine andPEG-nalbuphine conjugates was measured using radioligand binding assaysin CHO cells that heterologously express the recombinant human mu, deltaor the kappa opioid receptor. Cells were plated in 24 well plates at adensity of 0.2-0.3*10⁶ cells/well and washed with assay buffercontaining 50 mM Tris.HCl and 5 mM MgCl₂ (pH 7.4). Competition bindingassays were conducted in whole cells incubated with increasingconcentrations of test compounds in the presence of appropriateconcentration of radioligand. 0.5 nM ³H Naloxone, 0.5 nM ³HDiprenorphine and 0.5 nM ³H DPDPE were used as the competingradioligands for mu, kappa and delta receptors respectively. Incubationswere carried out for two hours at room temperature using triplicatewells at each concentration. At the end of the incubation, cells werewashed with 50 mM Tris HCl (pH 8.0), solubilized with NaOH and boundradioactivity was measured using a scintillation counter.

Specific binding is determined by subtraction of the cpm bound in thepresence of 50-100× excess of cold ligand. Binding data assays wereanalyzed using GraphPad Prism 4.0 and IC50 is generated by non-linearregression from dose-response curves. Ki values were calculated usingthe Cheng Prusoff equation using the Kd values from saturation isothermsas follows: Ki=IC50/(1+[Ligand]/Kd).

PEG conjugates of nalbuphine retain binding affinity to opioidreceptors. Table 1 shows the binding affinity (Ki, in nM) for PEGconjugates of nalbuphine at the mu, delta and kappa opioid receptors.The loss in binding affinity following PEG conjugation is less than15-fold that of parent nalbuphine at all three receptor subtypes. SeeTable 1. PEG conjugation results in a 10-15 fold loss in bindingaffinity at the mu and kappa opioid receptors, but not at the deltaopioid receptors. Binding affinity at the delta opioid receptor iscomparable, or even greater in some cases, than that of parentnalbuphine. See FIG. 1. The differential change in binding affinity atthe three opioid receptor subtypes implies that the receptor selectivityof the nalbuphine conjugates is altered compared to the parentnalbuphine.

TABLE 1 Binding Activities Ki at KAPPA Fold vs Ki at MU Fold vs Ki atDELTA Fold vs receptors Nalbuphine Receptors Nalbuphine ReceptorsNalbuphine Molecule (nM) at KAPPA (nM) At MU (nM) At DELTA Nalbuphine25.95 1 14.31 1 318.85 1 3-O-mPEG_(n)- — — — — — — Nalbuphine*6-O-mPEG₃- 218.1 8.40 27.41 1.92 163.30 0.51 Nalbuphine 6-O-mPEG₄- 17.541.23 148.90 0.47 Nalbuphine 6-O-mPEG₅- 35.56 1.37 35.09 2.45 147.70 0.46Nalbuphine 6-O-mPEG₆- 246.9 9.51 44.28 3.09 130.00 0.41 Nalbuphine6-O-mPEG₇- 346.1 13.34 77.94 5.45 313.80 0.98 Nalbuphine 6-O-mPEG₈-282.2 10.87 79.55 5.56 167.50 0.53 Nalbuphine 6-O-mPEG₉- 186.1 7.17122.30 8.55 157.70 0.49 Nalbuphine *The “3-O-mPEG_(n)-nalbuphine” seriesof molecules prepared in Example 1 showed no detectable bindingactivity; molecules wherein a water-soluble, non-peptidic oligomer iscovalently attached at the 3-O position are believed to have value when,for example, the covalent linkage is a degradable form of linkage. Somebinding activity values in the above table have been replaced withvalues obtained under further optimized assay conditions. Although theoriginal values are believed to be reliable and useful, they have beenreplaced here in the interest of brevity.

Example 5 Preparation of U50488 Conjugates

PEG-U50488 can be prepared following the approaches schematically shownbelow.

Example 5A Synthesis of mPEG_(n)-O-U50488 Conjugates

Following the general schematic provided below, mPEG_(n)-O-U504488conjugates can be prepared.

(+/−)-trans-2-(Pyrrolidin-1-yl)cyclohexanol ((+/−)-1)

Pyrrolidine (4.26 g, 60 mmol) was added to a solution of cyclohexeneoxide (1.96 g, 20 mmol) in H₂O (6 mL), and the resulting mixture washeated at 90° C. for 16 hours. After cooling, the solvent was removedunder reduced pressure and the resulting residue was extracted with DCM(10 mL×3). The organic phasew were combined and dried with anhydrousNa₂SO₄. After removing the Na₂SO₄ by filtration, the solvent wasevaporated and the material dried under vacuum to afford the desiredcompound (+/−)-1 (3.3 g, 19.5 mmol, yield 98%). ¹H NMR (CDCl₃) δ 4.06(s, 1H), 3.45-3.30 (m, 1H), 2.75-2.63 (m, 2H), 2.62-2.50 (m, 2H),2.51-2.42 (m, 1H), 1.90-1.58 (m, 8H), 1.45-1.15 (m, 4H).

(+/−)-trans-N-Methyl-2-(pyrrolidin-1-yl)cyclohexanamine ((+/−)-2)

To a solution of compound (+/−)-1 (1.01 g, 6 mmol) and TEA (0.98 mL, 7mmol) in DCM (20 mL) was added methanesulfonyl chloride (0.51 mL, 6.6mmol) dropwise. The reaction mixture was stirred for 2 h at roomtemperature. The solvent was removed under reduced pressure, and 5 mLmethylamine (40% in H₂O) was added at room temperature. The solution wasstirred at room temperature for an additional 16 h. The reaction mixturewas then added to DCM (50 mL) and washed with sat. NaHCO₃ solution (2×25mL). The organic phases were combined and dried with anhydrous Na₂SO₄.After removing the Na₂SO₄ by filtration, the solvent was removed and thematerial dried under vacuum to afford the desired compound (+/−)-2 (1.0g, 5.5 mmol, yield 91%). ¹H NMR (CDCl₃) δ 2.61 (s, 1H), 2.49-2.30 (m,6H), 2.29 (s, 3H), 2.09-1.96 (m, 2H), 1.67-1.52 (m, 7H), 1.11-1.02 (m,3H).

(+/−)-N-methyl-N-[2-(pyrrolidin-1-yl)cyclohexyl]-2-(3-chloro-4-hydroxyphenyl)acetamide((+/−)-4)

3-Chloro-4-hydroxyphenylacetic acid (186 mg, 1 mmol) and NHS (115 mg, 3mmol) were dissolved in DCM (20 mL). Then DCC (1.1 mmol) was added intothe solution and the reaction mixture was stirred at room temperaturefor 16 h during which a precipitate was formed. After the precipitatewas removed by filtration, the resulting filtrate was mixed withcompound (+/−)3 (182 mg, 1 mmol). The resulting solution was stirred atroom temperature overnight. The solvent was removed under reducedpressure and the resulting residue was subjected to flash chromatographypurification (MeOH/DCM=2%˜18%) to give the desired product (+/−)-4 (120mg, 0.34 mmol, yield 34%). ¹H NMR (CDCl₃) 7.12 (s, 1H), 7.00-6.86 (m,2H), 4.56 (s, 1H), 3.66-3.61 (m. 2H), 3.09-2.85 (m, 6H), 2.85 (s, 3H),1.71-1.45 (m, 8H), 1.44-1.11 (m 4H). LC/MS 351.1 [M+H]⁺.

(+/−)-N-methyl-N-[2-(pyrrolidin-1-yl)cyclohexyl]-2-(3-chloro-4-methoxy-tri(ethyleneglycol)phenyl)acetamide ((+/−)-5a)

Compound (+/−)-4 (50 mg, 0.14 mmol), methoxy tri(ethylene glycol)methanesulfonate (48.4 mg, 0.2 mmol) and anhydrous K₂CO₃ (70 mg, 0.5mmol) were added to acetone (10 mL). The resulting mixture was stirredunder reflux for 16 h. The solid was removed by filtration and thesolvent was evaporated under reduced pressure. The resulting residue wassubjected to flash chromatography purification (MeOH/DCM=2%˜15%) to givethe desired compound (+/−)-5a (30 mg, 0.06 mmol, yield 43%). ¹H NMR(CDCl₃) δ 7.31 (s, 1H), 7.18 (d, 1H), 7.06 (d, 1H), 4.61-4.51 (m 1H),4.19 (t, 2H), 3.90 (t, 2H), 3.76-3.44 (m, 10H), 3.36 (s, 3H), 2.95-2.83(m, 6H), 2.11-1.19 (m, 12H). LC/MS 497.2 [M+H]⁺

(+/−)-N-methyl-N-[2-(pyrrolidin-1-yl)cyclohexyl]-2-(3-chloro-4-methoxy-penta(ethyleneglycol)phenyl)acetamide ((+/−)-5b)

Compound (+/−)-4 (80 mg, 0.23 mmol), methoxy penta(ethylene glycol)methanesulfonate (108.9 mg, 0.33 mmol) and anhydrous K₂CO₃ (112 mg, 0.8mmol) were added to acetone (15 mL). The mixture was stirred underreflux for 16 h. The solid was removed by filtration and the solvent wasevaporated under reduced pressure. The resulting residue was subjectedto flash chromatography purification (MeOH/DCM=2%˜15%) to give thedesired compound (+/−)-5a (60 mg, 0.10 mmol, yield 45%). ¹H NMR (CDCl₃)δ 7.25 (s, 1H), 7.10 (d, 1H), 6.85 (d, 1H), 4.55-4.45 (m 1H), 4.14 (t,2H), 3.87 (t. 2H), 3.81-3.42 (m, 18H), 3.37 (s, 3H), 2.90-2.35 (m, 6H).2.09-1.15 (m, 12H). LC/MS 585.3 [M+H]⁺.

(+/−)-N-methyl-N-[2-(pyrrolidin-1-yl)cyclohexyl]-2-(3-chloro-4-methoxy-hexa(ethyleneglycol)phenyl)acetamide ((+/−)-5c)

Compound (+/−)-4 (50 mg, 0.23 mmol), methoxy hexa(ethylene glycol)methanesulfonate (150 mg, 0.37 mmol) and anhydrous K₂CO₃ (112 mg, 0.8mmol) were added to acetone (15 mL). The mixture was stirred underreflux for 16 h. The solid was removed by filtration and the solvent wasevaporated under reduced pressure. The resulting residue was subjectedto flash chromatography purification (MeOH/DCM=2%˜15%) to give thedesired compound (+/−)-5c (61 mg, 0.09 mmol, yield 39%). ¹H NMR (CDCl₃)δ 7.27 (s, 1H), 7.16 (d, 1H), 6.88 (d, 1H), 4.72-4.51 (m 1H), 4.16 (t,2H), 3.89 (t, 2H), 3.79-3.49 (m, 26H), 3.38 (s, 3H), 3.18-2.53 (m, 6H),2.10-1.12 (m, 12H). LC/MS 673.4 [M+H]⁺.

Example 5B Synthesis of di-mPEG_(n)-CH₂—U50488 Conjugates

Following the general schematic provided below, di-mPEG_(n)-CH₂-U504488conjugates can be prepared.

3-Cyclohexene 1,1-dimethanol methoxy tri(ethylene glycol) ether (2a)

3-Cyclohexene 1,1-dimethanol 1 (284 mg, 2 mmol) was dissolved inanhydrous DMF (6 mL). At room temperature, NaH (60% in mineral oil, 320mg, 8 mmol) was added and the solution was stirred for an additional for10 minutes. Methoxy tri(ethylene glycol) mesylate (1.21 g, 5 mmol) wasadded to the solution. The reaction solution was stirred at 45° C. for18 h and then saturated NH₄Cl solution (30 mL) was added to thesolution. The solution was extracted with EtOAc (20 mL×2). The organicphases were combined, dried with Na₂SO₄, filtered and the solventremoved under reduced pressure to give compound 2a (850 mg, 1.96 mmol,98% yield). ¹H NMR (CDCl₃) δ 5.62-5.58 (m, 2H), 3.66-3.51 (m, 24H), 3.38(s, 6H), 3.34 (d, 2H). 3.25 (d, 2H), 2.01 (m, 2H), 1.87 (m, 2H), 1.52(t, 2H). LC/MS 435 [M+H]⁺, 452 [M+NH₄]⁺, 457 [M+Na]⁺.

3-Cyclohexene 1,1-dimethanol methoxy di(ethylene glycol) ether (2b)

3-Cyclohexene 1,1-dimethanol (426 mg, 3 mmol) 1 was dissolved inanhydrous DMF (9 mL). At room temperature, NaH (60% in mineral oil, 480mg, 12 mmol) was added and the solution was stirred for an additional 10minutes. Methoxy di(ethylene glycol) mesylate (1.5 g, 7.5 mmol) wasadded to the solution. The reaction solution was stirred at 45° C. for18 h and then saturated NH₄Cl solution (30 mL) was added to thesolution. The solution was extracted with EtOAc (20 mL×2). The organicphases were combined, dried with Na₂SO₄, filtered, and the solventremoved under reduced pressure to give compound 2b (1.18 g, 2.9 mmol,98% yield). ¹H NMR (CDCl₃) δ 5.64-5.56 (m, 2H), 3.66-3.54 (m, 16H), 3.35(s, 6H), 3.33 (d, 2H), 3.28 (d, 2H), 1.99 (m, 2H), 1.87 (m, 2H), 1.53(t, 2H).

3,3-Di[methoxy tri(ethylene glycol)methyl]-7-oxabicyclo[4.1.0]heptanes(3a)

Compound 2a (850 mg, 1.96 mmol) was dissolved in DCM (20 mL). At roomtemperature, mCPBA (77% max, 0.75 g, ˜3 mmol) was added to the solution.The reaction mixture was stirred at room temperature for 3.5 h. Sat.Na₂S₂O₃ solution (10 mL) was added to the solution and stirring occurredfor an additional 10 min. The resulting solution was extracted with DCM(20 mL×2). The organic phases were combined, dried with Na₂SO₄,filtered, and the solvent removed under reduced pressure to givecompound 3a (870 m g, 1.86 mmol. 95% yield). ¹H NMR (CDCl₃) δ 3.66-3.55(m, 24H), 3.38 (s, 6H), 3.27-3.12 (m, 6H), 1.99-1.67 (m, 6H). LC/MS 451[M+H]⁺, 468 [M+NH₄]⁺, 473 [M+Na]⁺.

3,3-Di[methoxy di(ethylene glycol)methyl]-7-oxabicyclo[4.1.0]heptanes(3b)

Compound 2b (1.18 g, 3.41 mmol) was dissolved in DCM (20 mL). At roomtemperature, mCPBA (77% max, 1.3 g, ˜5.2 mmol) was added to thesolution. The reaction solution was stirred at room temperature for 3.5h. Sat. Na₂S₂O₃ solution (15 mL) was added to the solution and stirringoccurred for an additional 10 min. The resulting solution was extractedwith DCM (20 mL×2). The organic phases were combined, dried with Na₂SO₄,filtered, and the solvent removed under reduced pressure to givecompound 3b (1.27 g, 3.12 mmol, 92% yield). ¹H NMR (CDCl₃) δ 3.65-3.54(m, 24H), 3.38 (s, 6H), 3.27-3.10 (m, 6H), 2.00-1.68 (m, 6H).

Trans-(+/−)-4 or -5-di[methoxy tri(ethyleneglycol)methyl]-2-(1-pyrrolidinyl)-cyclohexanol (4a)

Compound 3a (870 mg, 1.93 mmol) and pyrrolidine (2.5 mL) were added towater (8 mL) and heated to reflux for 5 h. The resulting solution wasextracted with DCM (10 mL×2). The organic phases were combined, driedwith Na₂SO₄, filtered, and the solvent removed under reduced pressure togive 4a as a mixture of 4ax and 4ay (910 mg total). The product was usedin the next reaction without further purification.

Trans-(+/−)-4 or -5-di[methoxy di(ethyleneglycol)methyl]-2-(1-pyrrolidinyl)-cyclohexanol (4b)

Compound 3b (1.27 g, 3.12 mmol) and pyrrolidine (2.5 mL) were added towater (8 mL) and heated to reflux for 5 h. The resulting solution wasextracted with DCM (10 mL×2). The organic phases were combined, driedwith Na₂SO₄, filtered, and the solvent removed under reduced pressure togive 4b as a mixture of 4bx and 4by (1.3 g total). The product was usedin the next reaction without further purification.

Trans-(+/−)-4 or -5-di[methoxy tri(ethyleneglycol)methyl]-N-methyl-2-(1-pyrrolidinyl)-cyclohexanamine (5a)

Compound 4a (910 mg, 1.74 mmol) was dissolved in DCM (20 mL), and TEA(0.42 mL, 3 mmol) was added. At room temperature, methanesulfonylchloride (0.16 mL, 2 mmol) was added dropwise. After stirring wascontinued overnight at room temperature, the resulting mixture wasevaporated under reduced pressure. The resulting residue was dissolvedin methylamine (40% w/w in water, 6 mL) and the solution was stirred atroom temperature for 30 h. The solution was then extracted with DCM (10mL×2). The organic phases were combined, dried with Na₂SO₄, filtered,and the solvent removed under reduced pressure to give 5a as a mixtureof 5ax and 5ay. The product was used in the next reaction withoutfurther purification.

Trans-(+/−)-4 or -5-di[methoxy di(ethyleneglycol)methyl]-N-methyl-2-(1-pyrrolidinyl)-cyclohexanamine (5b)

Compound 4b (1.3 g, 3 mmol) was dissolved in DCM (20 mL), and TEA (0.84mL, 6 mmol) was added. At room temperature, methanesulfonyl chloride(0.28 mL, 3.5 mmol) was added dropwise. After stirring was continuedovernight at room temperature, the resulting mixture was evaporatedunder reduced pressure. The obtained residue was dissolved inmethylamine (40% w/w in water, 6 mL) and the solution was stirred atroom temperature for 30 h. The resulting solution was then extractedwith DCM (10 mL×2). The organic phases were combined, dried with Na₂SO₄,filtered, and the solvent removed under reduced pressure to give 5b as amixture of 5bx and 5by. The product was used in the next reactionwithout further purification.

Trans-(+/−)-4 or -5-di[methoxy tri(ethyleneglycol)methyl]-N-methyl-N-[2-(pyrrolidin-1-yl)cyclohexyl]-2-(3,4-dichloro)acetamide(6a)

3,4-Dichlorophenylacetic acid (410 mg, 2 mmol), compound 5a (926 mg,1.74 mmol), and DMAP (10 mg) were dissolved in DCM (10 mL). Then DCC(515 mg, 2.5 mmol) was added to the solution and the reaction mixturewas stirred at room temperature for 4 h during which a precipitateformed. After the precipitate was removed by filtration, the resultingfiltrate solvent was evaporated and the residue was subjected to flashchromatography purification (MeOH/DCM=2%˜8%) to give the desired product6a as a mixture of 6ax and 6ay (477 mg total, 0.34 mmol, yield 20%). ¹HNMR (CDCl3) δ 7.39-7.33 (m, 2H), 7.15-7.10 (m, 1H), 3.71-3.51 (m, 28H),3.42-3.16 (m, 3H), 3.35 (s, 6H), 2.82, 2.79 (s, s, 3H total, ratio 3:5),2.70-2.30 (m, 5H), 1.73-1.17 (m, 11H). LC/MS 721 [M+H]⁺, 743 [M+Na]⁺.

Trans-(+/−)-4 or -5-di[methoxy di(ethyleneglycol)methyl]-N-methyl-N-[2-(pyrrolidin-1-yl)cyclohexyl]-2-(3,4-dichloro)acetamide(6b)

3,4-Dichlorophenylacetic acid (707 mg, 3.45 mmol), compound 5b (1.33 g,2.98 mmol), and DMAP (10 mg) were dissolved in DCM (10 mL). Then DCC(865 mg, 4.2 mmol) was added to the solution and the reaction mixturewas stirred at room temperature for 4 h during which a precipitate wasformed. After the precipitate was removed by filtration, the resultingfiltrate solvent was evaporated and the residue was subjected to flashchromatography purification (MeOH/DCM=2%˜8%) to give the desired product6b as a mixture of 6bx and 6by (306 mg total, 0.49 mmol, yield 16%). ¹HNMR (CDCl3) δ 7.33-7.27 (m, 2H), 7.06-7.04 (m, 1H), 3.65-3.46 (m, 20H),3.42-3.12 (m, 3H), 3.37 (s, 6H), 2.76, 2.74 (s, s, 3H total, ratio1.1:1), 2.72-2.24 (m, 5H), 1.71-1.07 (m, 11H). LC/MS 633 [M+H]⁺, 655[M+Na]⁺.

Example 6 Preparation of an Oligomer-U69593 Conjugates

PEG-U69593 can be prepared following the approach schematically shownbelow. Conventional organic synthetic techniques are used in carryingout the approach.

Example 7 Preparation of Conjugates Other than with Nalbuphine, U50488,and U69593

Conjugates of opioid agonists other than nalbuphine, U50488 and U69593can be prepared wherein the general synthetic scheme and procedures setforth in Example 1 can be followed except that an opioid agonist ofFormula I is substituted for nalbuphine, U50488 and U69593.

Example 8 In Vitro Efficacy of PEG-Nalbuphine Conjugates

The in vitro efficacy of PEG-nalbuphine conjugates was determined usinga GTPγS binding assay in CHO cells expressing the recombinant human muor delta opioid receptors or HEK cells expressing the recombinant humankappa opioid receptor. Test compound and/or vehicle was preincubatedwith the cell membranes and 3 μM GDP in modified HEPES buffer (pH 7.4)for 20 minutes, followed by addition of SPA beads for another 60 minutesat 30° C. The reaction is initiated by 0.3 nM [³⁵S]GTPγS for anadditional 30 minutes incubation period. Test compound-induced increaseof [³⁵S]GTPγS binding by 50 percent or more (≧50%) relative to thereceptor subtype-specific agonist response indicates possible opiatereceptor agonist activity. 0.1 μM DPDPE, 1 μM U-69593 and 1 μM DAMGOwere used as the specific agonists for the delta, kappa and mu opioidreceptors respectively. Opioid receptor antagonist activity was measuredusing inhibition of agonist-induced increase of [³⁵S]GTPγS bindingresponse by 50 percent or more (≧50%). Nalbuphine, 6-O-mPEG₃-Nalbuphine,6-O-mPEG₆-Nalbuphine, 6-O-mPEG₉-Nalbuphine were screened atconcentrations of 10, 1, 0.1, 0.01 and 0.001 μM in both agonist andantagonist mode. EC₅₀ or IC₅₀ values were calculated from thedose-response curves as a measure of the agonist or antagonist activityof the test compounds respectively.

Table 2 shows the EC₅₀/IC₅₀ values of nalbuphine and PEG-nalbuphineconjugates to activate or inhibit GTPγS binding, thus reflecting theiragonist or antagonist activity. PEG-nalbuphine conjugates are fullagonists at kappa opioid receptors and antagonists at mu opioidreceptors, similar to the pharmacological profile of nalbuphine. TheEC₅₀ of 6-O-mPEG₃-Nalbuphine was similar to that of nalbuphine at thekappa opioid receptor, suggesting no loss of efficacy at this PEG size.Beyond a PEG size of 3, the efficacy of the PEG-nalbuphine conjugates atthe kappa opioid receptor decreased as a function of PEG size, asindicated by the increase in EC₅₀ values of 6-O-mPEG₆-Nalbuphine, and6-O-mPEG₉-Nalbuphine. PEG-nalbuphines appeared to have an antagonistpotency at the mu opioid receptor comparable to that of the parentnalbuphine. At the delta opioid receptor, 6-O-mPEG₉-Nalbuphine, acted asa weak antagonistic, however, nalbuphine, 6-O-mPEG₃-Nalbuphine,6-O-mPEG₆-Nalbuphine, had no effect at the delta opioid receptor.

TABLE 2 PEG-nalbuphine Conjugates Retain the in vitro PharmacologicalProperties of Parent Nabuphine Functional Assay GTP-γS Binding Kappa MuAgonist Antagonist Delta Molecule EC50 (nM) IC50 (nM) IC50 (nM)Nalbuphine 25.1 752 6-O-mPEG₃-Nalbuphine 26.80 398.0 —6-O-mPEG₆-Nalbuphine 164.00 284.0 — 6-O-mPEG₉-Nalbuphine 485.00 1010.08970.0

Example 9 In Vitro Permeability

The in vitro permeability of PEG-nalbuphine conjugates was measuredusing a bi-directional transport assay in Caco-2 cells. PEG-nalbuphineconjugates were added at a concentration of 10 μM to either the apicalor basolateral compartment of Caco-2 monolayers and incubated for twohours. At the end of the incubation, the concentrations in the apicaland basolateral compartments were measured using LC-MS. Permeability wascalculated as Papp=J/A.Co, where Papp is the apparent permeability incm/s, J is the flux in moles/s, A is the surface area of the insert incm² and Co is the initial concentration in moles/cm³.

FIG. 2A shows the in vitro apparent permeability of PEG-nalbuphineconjugates measured in Caco-2 cells in the A-B (apical-basolateral) andB-A (basolateral-apical) direction. A-B permeability, and to a lesserextent, B-A permeability decrease with increasing PEG chain length. TheA-B permeability, which represents mucosal-serosal transport in vivo isgreater than 1*10⁻⁶ cm/s for all compounds, indicating that nalbuphineand PEG-nalbuphine conjugates are likely to be well absorbed orally.

FIG. 2B shows the efflux ratio of PEG-nalbuphine conjugates, calculatedas a ratio of B-A/A-B permeabilities. An efflux ratio greater than unityis a reflection of an asymmetry in transport in the apical-basolateraldirections and suggests a role for transporters in the overallpermeability. The efflux ratio for the parent nalbuphine is close tounity, indicating that it is not a likely substrate for transporters.However, 6-O-mPEG₅-Nalbuphine, 6-O-mPEG₆-Nalbuphine.6-O-mPEG₇-Nalbuphine, 6-O-mPEG₅-Nalbuphine, and 6-O-mPEG₉-Nalbuphinehave an efflux ratio greater than 3, and hence are likely substrates forefflux transporters in Caco-2 cells.

Example 10 In Vivo Brain Penetration of PEG-Nalbuphine Conjugates

The ability of the PEG-nalbuphine conjugates to cross the blood brainbarrier (BBB) and enter the CNS was measured using the brain:plasmaratio in rats. Briefly, rats were injected intravenously with 25 mg/kgof nalbuphine, PEG-nalbuphine conjugate or atenolol. An hour followinginjection, the animals were sacrificed and plasma and the brain werecollected and frozen immediately. Following tissue and plasmaextractions, concentrations of the compounds in brain and plasma weremeasured using LC-MS/MS. The brain:plasma ratio was calculated as theratio of measured concentrations in the brain and plasma. Atenolol,which does not cross the BBB was used as a measure of vascularcontamination of the brain tissue.

FIG. 3 shows the ratio of brain:plasma concentrations of PEG-nalbuphineconjugates. The brain:plasma ratio of nalbuphine was 2.86:1, indicatinga nearly 3 fold greater concentration of nalbuphine in the braincompared to the plasma compartment. PEG conjugation significantlyreduced the entry of nalbuphine into the CNS as evidenced by a lowerbrain:plasma ratio of the PEG-nalbuphine conjugates. Conjugation with 3PEG units reduced the brain:plasma ratio to 0.23:1, indicating that theconcentration of 6-O-mPEG₃-Nalbuphine in brain was approximately 4 foldless than that in the plasma. 6-O-mPEG₆-Nalbuphine and6-O-mPEG₉-Nalbuphine were significantly excluded from the CNS, sincetheir brain:plasma ratios were not significantly different from thevascular marker, atenolol.

Example 11 Preparation of Oligomer-Fentanyl Conjugates

mPEG_(n)-O-fentanyl conjugates can be prepared following the approachesschematically shown below. Conventional organic synthetic techniques areused in carrying out the synthetic approaches.

An exemplary approach for preparing the following structures, where thePEG oligomer is positioned, i.e., covalently attached, at theN-(1-(2-phenylethyl)piperidin-4-yl)phenyl group:

[wherein mPEG_(n) is —(CH₂CH₂O)_(n)—CH₃ and n is an integer from 1 to9],

is provided below.

In the above approach, the starting material is a(haloethyl)hydroxybenzene, where the hydroxy group forms the point ofattachment for the PEG oligomer. The (haloethyl)hydroxybenzene, i.e.,(bromoethyl)hydroxybenzene, is reacted with a mesylated or halogenatedactivated mPEG oligomer, thereby forming the desired PEG-oligomermodified (haloethyl)benzene intermediate. This intermediate is thenreacted with piperidin-4-one in the presence of a phase transfercatalyst; the bromo group reacts at the piperidine-4-one nitrogen toform a next intermediate, 1-(mPEG_(olig)-phenylethyl)piperidine-4-one.The ketone functionality is then reduced in the presence of a reducingagent such as sodium borohydride, and converted to an amino group, i.e.,N-phenyl-piperidin-4-amine, by reaction with aniline. Finally, thesecondary amino group is converted to a tertiary amine by reaction withpropionyl chloride to form the desired product as indicated in thescheme above.

The subject mPEG_(n)-O-fentanyl conjugates having the PEG oligomerpositioned at the N-(1-(2-phenylethyl)piperidin-4-yl)phenyl group weresynthesized using a reaction scheme that was slightly modified fromScheme 11-A above as illustrated in Scheme 11-B below:

The above approach employs tosyl (p-toluenesulfonate) leaving groups atvarious steps in the synthesis. The desired PEG-oligomer conjugates (n=1to 9) were assembled by reacting di-tosylated 3-(2-hydroxyethyl)phenolwith N-phenyl-N-(piperidin-4-yl)propionamide to formN-(1-(3-hydroxyphenylethyl)piperidin-4-yl)-N-phenylpropionamide, intosylated form, followed by removal of the tosyl group. The PEG-oligomergroup was then introduced into the molecules at the phenyl hydroxylposition by reaction ofN-(1-(3-hydroxyphenylethyl)piperidin-4-yl)-N-phenylpropionamide with amolar excess of mPEG_(olig)-tosylate to form the desiredmPEG_(n)-O-fentanyl conjugates. Ratios of reactants and reactionconditions generally employed are provided in the reaction scheme above.

An exemplary approach for providing the following structures, where thePEG oligomer is positioned, i.e., covalently attached, at the N-phenylgroup, is set forth below:

The above exemplary approach for forming an mPEG_(n)-O-fentanylconjugate having the PEG oligomer positioned at the N-phenyl ring startswith, e.g., 2-bromoethylbenzene, as the starting material. The2-bromoethylbenzene is reacted with piperidin-4-one in the presence of aphase transfer catalyst to thereby form the resulting1-phenethylpiperidin-4-one. The 1-phenethylpiperidin-4-one is coupled tomPEG_(olig)-substituted aniline, which is prepared by taking N-protectedhydroxyaniline and reacting it with activated mPEG oligomer, such asbromomethoxyPEG_(olig) or mPEG_(oligo) mesylate, followed by removal ofthe protecting group (see step (b) above). As indicated in reaction step(c) above, 1-phenethylpiperidin-4-one is reacted withmPEG_(olig)-substituted aniline in the presence of a reducing agent toconvert the keto group into an amine to form the intermediate,1-phenylethylpiperidin-4-ylamino-mPEG_(olig)obenzene. Finally, thesecondary amino group is converted to a tertiary amine by reaction withpropionyl chloride to form the desired product as indicated in thescheme above.

The subject mPEG_(n)-O′-fentanyl conjugates having the PEG oligomerpositioned at the N-phenyl group were synthesized using a reactionscheme that was slightly modified from Scheme 11-C above as illustratedin Scheme 11-D below:

As indicated in Scheme 11-D above, the desired mPEG_(n)-O-fentanylconjugates were prepared by first reacting 1-phenethylpiperidin-4-onewith 3-aminophenol under reducing conditions to thereby convert the ketofunctionality into an amine, i.e., by reaction with the amino group of3-aminophenol. The product, 3-(1-phenethylpiperidin-4-ylamino)phenol,was then reacted with propionic anhydride in the presence of base (e.g.,triethyl amine) and dimethylaminopyridine (DMAP) under conditionseffective to formN-(3-hydroxyphenyl)-N-(1-phenethylpiperidin-4-yl)propionamide. Finally,introduction of the oligomeric PEG functionality was carried out byreacting the precursor,N-(3-hydroxyphenyl)-N-(1-phenethylpiperidin-4-yl)propionamide, with amolar excess of mPEG_(oligo)tosylate under coupling conditions effectiveto form the desired conjugates. Ratios of reactants and reactionconditions generally employed are provided in the reaction schemesabove.

Example 11A Preparation of m-mPEG_(n)-O-Fentanyl Conjugates Synthesis ofm-mPEG₁-O-Fentanyl Conjugate (n=1)

Using an approach set forth in Example 11 and as described schematicallyin Scheme 11-B, the above conjugate was prepared.

Synthesis of m-mPEG₂-O-Fentanyl Conjugate (n=2)

Using an approach set forth in Example 11 and as described schematicallyin Scheme 11-B, the above conjugate was prepared.

Synthesis of m-mPEG₃-O-Fentanyl Conjugate (n=3)

Using an approach set forth in Example 11 and as described schematicallyin Scheme 11-B, the above conjugate was prepared.

Synthesis of m-mPEG₄-O-Fentanyl Conjugate (n=4)

Using an approach set forth in Example 11 and as described schematicallyin Scheme 11-B, the above conjugate was prepared.

Synthesis of m-mPEG₅-O-Fentanyl Conjugate (n=5)

Using an approach set forth in Example 11 and as described schematicallyin Scheme 11-B, the above conjugate was prepared.

Synthesis of m-mPEG₆-O-Fentanyl Conjugate (n=6)

Using an approach set forth in Example 11 and as described schematicallyin Scheme 11-B, the above conjugate was prepared.

Synthesis of m-mPEG₇-O-Fentanyl Conjugate (n=7)

Using an approach set forth in Example 11 and as described schematicallyin Scheme 11-B, the above conjugate was prepared.

Synthesis of m-mPEG₇-O-Fentanyl Conjugate (n=7)

Using a similar approach set forth in Example 11 and as describedschematically in Scheme 11-B, the above conjugate was prepared.

Synthesis of m-mPEG₈-O-Fentanyl Conjugate (n=8

Using an approach set forth in Example 11 and as described schematicallyin Scheme 11-B, the above conjugate was prepared.

Synthesis of m-mPEG₉-O-Fentanyl Conjugate (n=9)

Using an approach set forth in Example 11 and as described schematicallyin Scheme 11-B, the above conjugate was prepared.

Each of the above mPEG₁₋₉-O-fentanyl conjugates was characterized by ¹HNMR (200 MHz Bruker) and by LC/MS.

Example 12 Preparation of m-mPEG_(n)-O′-Fentanyl Conjugates Synthesis ofm-mPEG₁-O′-Fentanyl Conjugate (n=1)

Using an approach set forth in Example 11 and as described schematicallyin Scheme 11-D, the above conjugate was prepared. In this series, theoligomeric mPEG was covalently attached at the meta-position of theN-phenyl group

Synthesis of m-mPEG₂-O′-Fentanyl Conjugate (n=2)

The above conjugate was prepared using the approach set forth in Example11 and as described schematically in Scheme 11-D.

Synthesis of m-mPEG₃-O′-Fentanyl Conjugate (n=3)

The above conjugate was prepared using the approach set forth in Example11 and as described schematically in Scheme 11-D.

Synthesis of m-mPEG₄-O′-Fentanyl Conjugate (n=4)

The above conjugate was prepared using the approach set forth in Example11 and as described schematically in Scheme 11-D.

Synthesis of m-mPEG₅-O′-Fentanyl Conjugate (n=5)

The above conjugate was prepared.

Synthesis of m-mPEG₆-O′-Fentanyl Conjugate (n=6)

The above conjugate was prepared using the approach set forth in Example11 and as described schematically in Scheme 11-D.

Synthesis of m-mPEG₇-O′-Fentanyl Conjugate (n=7)

The above conjugate was prepared using the approach set forth in Example11 and as described schematically in Scheme 11-D.

Synthesis of m-mPEG₈-O′-Fentanyl Conjugate (n=8)

The above conjugate was prepared using the approach set forth in Example11 and as described schematically in Scheme 11-D.

Synthesis of m-mPEG₈-O′-Fentanyl Conjugate (n=8)

The above conjugate was prepared using the approach set forth in Example11 and as described schematically in Scheme 11-D.

Synthesis of m-mPEG₉-O′-Fentanyl Conjugate (n=9)

The above conjugate was prepared using the approach set forth in Example11 and as described schematically in Scheme 11-D.

Each of the above mPEG₁₋₉-O′-fentanyl conjugates was characterized by ¹HNMR (200 MHz Bruker) and by LC/MS.

Example 13 Preparation of para-mPEG_(n)-O′-Fentanyl Conjugates Synthesisof p-mPEG₁-O′-Fentanyl Conjugate (n=1)

The above conjugate can be prepared using an approach set forth inExample 11. In this series, the oligomeric mPEG is covalently attachedat the para-position of the N-phenyl group.

Synthesis of p-mPEG₄-O′-Fentanyl Conjugate (n=4)

The para-substituted conjugate was prepared according to the reactionscheme shown below:

The desired pPEG₄-O-fentanyl conjugate was prepared by first reacting1-phenethylpiperidin-4-one with 4-aminophenol under reducing conditions(e.g., in the presence of a reducing agent such as NaBH(OAc)₃) tothereby convert the keto functionality into an amine, i.e., by reactionwith the amino group of 4-aminophenol. The product,4-(1-phenethylpiperidin-4-ylamino)phenol, was then reacted withpropionic anhydride in the presence of base (e.g., triethyl amine) anddimethylaminopyridine (DMAP) under conditions effective to formN-(4-hydroxyphenyl)-N-(1-phenethylpiperidin-4-yl)propionamide. Finally,introduction of the oligomeric PEG functionality was carried out byreacting the precursor,N-(4-hydroxyphenyl)-N-(1-phenethylpiperidin-4-yl)propionamide, with amPEG₄tosylate under coupling conditions effective to form the desiredconjugate. Ratios of reactants and reaction conditions generallyemployed are provided in the reaction scheme above.

Additional pPEG_(oligo)-O-fentanyl conjugates may be similarly prepared.

Example 14 Preparation of mPEG_(n)-OMs (mPEGn-O-Mesylate) for Use inExamples 15, 16 and 17

In a 40-mL glass vial was mixed HO—CH₂CH₂OCH₂CH₂—OH (1.2 ml, 10 mmol)and DIEA (N,N-diisopropylethylamine, 5.2 ml, 30 mmol, 3 eq), theresulting homogeneous colorless mixture was cooled to 0° C. and MsCl(1.55 ml, 20 mmol, 2 eq) was added via syringe slowly, over 4 minutes,with vigorous stirring. A biphasic mixture resulted upon addition:yellow solid on the bottom and clear supernatant. The ice bath wasremoved and the reaction was allowed to warm to room temperatureovernight. At this point it was dissolved in water, extracted with CHCl₃(3×50 mL), washed with 0.1 M HCl/brine mixture 2×50 mL, followed bybrine 50 mL. The organic layer was dried over MgSO₄, filtered to give ayellow solution and evaporated to give brown oil (2.14 g). ¹H NMRconfirms product identity 3.3 (1H NMR δ 3.1 (s, 3H), 3.3 (s, 3H),3.5-3.55 (m, 2H), 3.6-3.65 (m, 2H), 3.7-3.75 (m, 2H), 4.3-4.35 (m, 2H)ppm).

All other PEG_(n)-OMs's (n=3, 4, 5, 6, 7 and 9) were made in similarfashion and afforded final compounds in each case that were isolated asbrown oils. Mass spectral and proton NMR data (not shown) confirmed theformation of the desired OMs PEGylated products.

Example 15 Preparation of mPEG_(n)-O-Morphine Conjugates

The following describes the preparation of free base using commerciallyavailable morphine sulfate hydrate (generally procedure).

Morphine sulfate USP from Spectrum (510 mg) was dissolved in water (70ml). The solution was then basified to pH 10 using aqueous K₂CO₃ to givea white suspension. To the white suspension DCM (dichloromethane, 50 ml)was added, but failed to dissolve the solid. The mixture was made acidicwith 1M HCl to result in clear biphasic solution. The organic phase wassplit off and the aqueous phase was carefully brought to pH 9.30(monitored by a pH meter) using the same solution of K₂CO₃ as above. Awhite suspension resulted again. The heterogeneous mixture was extractedwith DCM (5×25 ml) and an insoluble white solid contaminated both theorganic and aqueous layers. The organic layer was dried with MgSO₄,filtered and rotary evaporated to yield 160 mg of morphine free base(56% recovery). No additional product was recovered from the filter cakeusing MeOH, but another 100 mg was recovered from the aqueous phase by2×50 ml extraction with EtOAc to give a combined yield of 260 mg (68%).

MEM Protection of Morphine Free Base

The general approach for protecting the free base of morphine with theprotecting group β-methoxyethoxymethyl ester (“MEM”) is schematicallyshown below.

Free base morphine (160 mg, 0.56 mmol) was dissolved in 20 ml ofAcetone/Toluene (2/1 mixture). To the resulting solution was added K₂CO₃(209 mg, 1.51 mmol, 2.7 eq) followed by MEMCl (96 d, 0.84 mmol, 1.5 eq)and the resulting heterogeneous mixture was stirred overnight at roomtemperature. After five hours at room temperature, the reaction wasdeemed complete by LC-MS. Morphine free base retention time understandard six minute gradient run conditions (std 6 min, Onyx MonolythC18 column, 50×4.6 mm; 0 to 100% Acetonitrile 0.1% TFA in Water 0.1%TFA, 1.5 ml/min; detection: UV254, ELSD, MS; retention times are quotedfor UV254 detector, ELSD has about 0.09 min delay and MS has about 0.04min delay relative to UV) was 1.09 min; retention time for product 1.54min (std 6 min), major impurity 1.79 min. The reaction mixture wasevaporated to dryness, dissolved in water, extracted with EtOAc (3×,combined organic layer washed with brine, dried over MgSO₄, filtered androtary evaporated) to give 160 mg (77%) of the desired product as acolorless oil. Product purity was estimated to be about 80% by UV254.

Direct MEM Protection of Morphine Sulfate (General Procedure)

The general approach for protecting morphine sulfate with the protectinggroup β-methoxyethoxymethyl ester (“MEM”) is schematically shown below.Although not explicitly shown in the scheme below, morphine is actuallymorphine sulfate hydrate, morphine. 0.5 H₂SO₄.2.5 H₂O.

To a suspension of 103 mg of morphine sulfate hydrate (0.26 mmol) in 10ml of 2:1 acetone:toluene solvent mixture was added 135 mg (1 mmol, 3.7eq) of K₂CO₃ and the suspension stirred at room temperature for 25minutes. To the resulting suspension was added 60 μl (0.52 mmol) ofMEMCl and the mixture allowed to react at room temperature. It wassampled after one hour (38% nominal conversion, additional peaks at 1.69min and 2.28 min), three hours (40% nominal conversion, additional peakat 1.72 min (M+1=493.2)), four and one-half hours (56% nominalconversion, additional peak at 1.73 min), and twenty-three hours (>99%nominal conversion, additional peak at 1.79 min—about 23% of the productpeak by height in UV₂₅₄); thereafter, the reaction was quenched withMeOH, evaporated, extracted with EtOAc to give 160 mg of clear oil.

The same reaction was repeated starting with 2 g (5.3 mmol) of morphinesulfate hydrate, 2.2 g (16 mmol, 3 eq) of K₂CO₃, 1.2 ml (10.5 mmol, 2eq) of MEMCl in 100 ml of solvent mixture. Sampling occurred after twohours (61% nominal conversion, extra peak at 1.72 min (M+1=492.8)),after one day (80% nominal conversion, extra peak at 1.73 min), afterthree days (85% nominal conversion, only small impurities, 12 mingradient run), and after six days (91% conversion); thereafter, thereaction was quenched, evaporated, extracted with EtOAc, purified oncombi-flash using a 40 g column, DCM:MeOH 0 to 30% mobile phase. Threepeaks (instead of two) were identified, wherein the middle peak wascollected, 1.15 g (58% yield) of light yellow oil, UV₂₅₄ purity about87%.

Conjugation of MEM-Protected Morphine to Provide a MEM-ProtectedMorphine Conjugate

The general approach for conjugating MEM-protected morphine with awater-soluble oligomer to provide a MEM-protected morphine PEG-oligomerconjugate is schematically shown below.

To a solution of toluene/DMF (2:1 mixture, 10 volumes total) was chargedMEM-morphine free base followed by NaH (4-6 eq) and then PEG_(n)OMs(1.2-1.4 eq.), previously prepared. The reaction mixture was heated to55-75° C. and was stirred until reaction completion was confirmed byLC-MS analysis (12-40 hours depending on PEG chain length). The reactionmixture was quenched with methanol (5 volumes) and the reaction mixturewas evaporated to dryness in vacuo. The residue was redissolved inmethanol (3 volumes) and was chromatographed using a Combiflash system(0-40% MeOH/DCM). The fractions containing large amounts of product werecollected, combined and evaporated to dryness. This material was thenpurified by RP-HPLC to give the products as yellow to orange oils.

Deprotection of MEM-Protected Morphine Conjugate to Provide a MorphineConjugate

The general approach for deprotecting a MEM-protected morphine conjugateto provide a morphine conjugate is schematically shown below.

To a solution of MEM-protected morphine conjugate TFA salt suspended inDCM (8 volumes) was charged 6 volumes of 2M HCl in diethyl ether. Thereaction mixture was allowed to stir at room temperature for two hoursand was then evaporated to dryness under reduced pressure. The oilyresidue was dissolved in MeOH (8 volumes), filtered through glass wooland then evaporated under reduced pressure to give a thick orange toyellow oil in quantitative yield. Compounds made by this method include:α-6-mPEG₃-O-morphine (Compound A, n=3) 217 mg of HCl salt 97% pure (95%by UV254; 98% by ELSD); α-6-mPEG₄-O-morphine (Compound A, n=4) 275 mg ofHCl salt 98% pure (97% by UV254; 98% by ELSD); α-6-mPEG₃-O-morphine(Compound A, n=5) 177 mg of HCl salt 95% pure (93% by UV254; 98% byELSD); α-6-mPEG₆-O-morphine (Compound A, n=6) 310 mg of HCl salt 98%pure (98% by UV254; 99% by ELSD); α-6-mPEG₇-O-morphine (Compound A, n=7)541 mg of HCl salt 96% pure (93% by UV254; 99% by ELSD); andα-6-mPEG-O₉-morphine (Compound A, n=9) 466 mg of HCl salt 98% pure (97%by UV254; 99% by ELSD). Additionally, morphine conjugates having asingle PEG monomer attached, α-6-mPEG₁-O-morphine (Compound A, n=1), 124mg of HCl salt, 97% pure (95% pure by UV₂₅₄; 98% by ELSD); as well asα-6-mPEG₂-O-morphine (Compound A, n=2), 485 mg of HCl salt, 97% pure(95% pure by UV₂₅₄; 98% by ELSD) were similarly prepared.

Example 16 Preparation of mPEG_(n)-O-Codeine Conjugates

The general approach for conjugating codeine with an activated sulfonateester of a water-soluble oligomer (using mPEG₃OMs as a representativeoligomer) to provide a codeine conjugate is schematically shown below.

Codeine (30 mg, 0.1 mmol) was dissolved in toluene/DMF (75:1) solventmixture followed by addition of HO—CH₂CH₂OCH₂CH₂OCH₂CH₂OMs (44 ml, 2 eq)and NaH (60% suspension in mineral oil, 24 mg, 6 eq). The resultinghomogeneous yellow solution was heated to 45° C. After one hour, thereaction showed 11% conversion (extra peak at 2.71 min, 12 min run),after eighteen hours, the reaction showed 7% conversion (extra peak at3.30 min, 12 min run), and after 24 hours, the reaction showed 24%conversion (multitude of extra peaks, two tallest ones are 1.11 min and2.79 min). At this point, an additional 16 mg of NaH was added andheating continued for six hours, after which, an additional 16 mg of NaHwas added followed by continued heating over sixty-six hours.Thereafter, no starting material remained, and analysis revealed manyextra peaks, the two tallest ones corresponding to 2.79 min and 3 min(product peak is the second tallest among at least 7 peaks).

This synthesis was repeated using 10× scale wherein 30 ml of solventmixture was used. After eighteen hours, analysis revealed 71% nominalconversion with additional peaks in the UV (one tall peak at 3.17 minand many small ones; wherein the desired peak corresponded to 3.43 minin UV). Thereafter, 80 mg (2 mmol) of NaH was added followed bycontinued heating. After three hours, analysis revealed 85% nominalconversion (several extra peaks, main 3.17 min). Reaction mixture wasdiluted with water, extracted with EtOAc (3×, combined organic layerwashed with brine, dried over MgSO₄, filtered and rotary evaporated) togive yellow oil (no sm in LC-MS, 90% pure by ELSD, 50% pure by UV—majorimpurity at 3.2 min). The crude product was dissolved in DCM, applied toa small cartridge filled with 230-400 mesh SiO₂, dried, eluted on aCombi-flash via a 4 g pre-packed column cartridge with solvent A=DCM andsolvent B=MeOH, gradient 0 to 30% of B. Analysis revealed two peaks ofpoor symmetry: a small leading peak and a larger peak with a tail. LC-MSwas used to analyze fractions, wherein none were identified ascontaining pure product. Combined fractions that contained any product(tt#22-30) yielded, following solvent evaporation, 150 mg (34% yield) ofimpure product (LC-MS purity at 3.35 min by UV254, wherein about 25%represented the main impurities 3.11 min, 3.92 min, 4.32 min, 5.61 minof a 12 min run). A second purification by HPLC (solvent A=water, 0.1%TFA; solvent B=acetonitrile, 0.1% TFA) employing a gradientcorresponding to 15-60% B, 70 min, 10 ml/min) resulted in poorseparation from adjacent peaks. Only two fractions were clean enough andgave 21 mg of TFA salt (>95% pure, 4.7% yield). Three additionalfractions both before and after the desired product-containing fractions(for a total of six additional fractions were combined to give 70 mg ofabout 50% pure product as TFA salts.

Using this same approach, other conjugates differing by the number ofethylene oxide units (n=4, 5, 6, 7, and 9) were made using these NaHconditions outlined above.

Conversion of Codeine-Oligomer Conjugate TFA Salts to Codeine-OligomerHCl Salts.

The general approach for converting codeine-oligomer TFA salts tocodeine-oligomer HCl salts is schematically shown below.

To a solution of codeine-oligomer conjugate TFA salt suspended in DCM (8volumes) was charged 6 volumes of 2M HCl in diethyl ether. The reactionmixture was allowed to stir at room temperature for two hours and wasthen evaporated to dryness under reduced pressure. The oily residue wasdissolved in MeOH (8 volumes), filtered through glass wool and thenevaporated under reduced pressure to give a thick orange to yellow oilin quantitative yield. Following this general procedure, the followingcompounds were synthesized: α-6-mPEG₃-O-codeine (Compound B, n=3) 235 mgof HCl salt. 98% pure; α-6-mPEG₄-O-codeine (Compound B, n=4) 524 mg ofHCl salt, 98% pure; α-6-mPEG₅-O-codeine (Compound B, n=5) 185 mg of HClsalt, 98% pure+119 mg of HCl salt 97% pure, α-6-mPEG₆-O-codeine(Compound B, n=6) 214 mg of HCl salt, 97% pure; α-6-mPEG₇-O-codeine(Compound B, n=7) 182 mg of HCl salt, 98% pure; α-6-mPEG₉-O-codeine(Compound B, n=9) 221 mg of HCl salt, 97% pure; α-6-mPEG₁-O-codeine(Compound B, n=1) 63 mg of HCl salt, 90% pure; and α-6-mPEG₂-O-codeine(Compound B, n=2) 178 mg of HCl salt, 90% pure.

Example 17 Preparation of mPEG_(n)-O-Hydroxycodone Conjugates

The general approach for conjugating hydroxycodone with an activatedsulfonate ester of a water-soluble oligomer (using “mPEG_(n)OMs” as arepresentative oligomer) to provide a hydroxycodone conjugate isschematically shown below.

Reduction of Oxycodone to α-6-hydroxycodone

To a solution of oxycodone free base in dry THF under nitrogen cooled at−20° C., was added a 1.0 M THF solution of potassiumtri-sec-butylborohydride over 15 minutes. The solution was stirred at−20° C. under nitrogen for 1.5 hours and then water (10 mL) was addedslowly. The reaction mixture was stirred another 10 minutes at −20° C.and then allowed to warm to room temperature. All solvents were removedunder reduced pressure and CH₂Cl₂ was added to the remaining residue.The CH₂Cl₂ phase was extracted with a 0.1 N HCl/NaCl water solution andthe combined 0.1 N HCl solution extracts were washed with CH₂Cl₂, thenNa₂CO₃ was added to adjust the pH=8. The solution was extracted withCH₂Cl₂. The CH₂Cl₂ extracts were dried over anhydrous Na₂SO₄. Afterremoving the solvent under reduced pressure, the desiredα-6-HO-3-hydroxycodone was obtained.

Conjugation of mPEG_(n)OMs to α-6-hydroxycodone

To a solution of Toluene/DMF (2:1 mixture, 10 volumes total) was chargedhydroxycodone (prepared as set forth in the preceding paragraph)followed by NaH (4 eq) and then mPEG_(n)OMs (1.3 e.). The reactionmixture was heated to 60-80° C. and was stirred until reactioncompletion was confirmed by LC-MS analysis (12-40 hours depending on PEGchain length). The reaction mixture was quenched with methanol (5volumes) and the reaction mixture was evaporated to dryness in vacuo.The residue was re-dissolved in methanol (3 volumes) and waschromatographed using Combiflash (0-40% MeOH/DCM). The fractionscontaining large amounts of product were collected, combined andevaporated to dryness. This material was then purified by RP-HPLC toprovide the final products as yellow to orange oils.

Conversion of Hydroxycodone Conjugate TFA Salts to HydroxycodoneConjugate HCl Salts

To a solution of hydroxycodone conjugate TFA salt suspended in DCM (8volumes) was charged 6 volumes of 2M HCl in diethyl ether. The reactionmixture was allowed to stir at room temperature for two hours and wasthen evaporated to dryness under reduced pressure. The oily residue wasdissolved in MeOH (8 volumes), filtered through glass wool and thenevaporated under reduced pressure to give a thick orange to yellow oilin quantitative yield. Following this general procedure, the followingcompounds were synthesized: α-6-mPEG₃-O-oxycodone (akaα-6-mPEG₃-O-hydroxycodone) (Compound C, n=3) 242 mg of HCl salt, 96%pure; α-6-mPEG₄-O-oxycodone (aka α-6-mPEG₄-O-hydroxycodone) (Compound C,n=4) 776 mg of HCl salt, 94% pure; α-6-mPEG₅-O-oxycodone (akaα-6-mPEG₅-O-hydroxycodone) (Compound C, n=5) 172 mg of HCl salt, 93%pure; α-6-mPEG₆-O-oxycodone (aka α-6-mPEG₆-O-hydroxycodone) (Compound C,n=6) 557 mg of HCl salt, 98% pure; α-6-mPEG₇-O-oxycodone (akaα-6-mPEG₇-O-hydroxycodone) (Compound C, n=7) 695 mg of HCl salt, 94%pure; and α-6-mPEG₉-O-oxycodone (aka α-6-mPEG₉-O-hydroxycodone)(Compound C, n=9) 435 mg of HCl salt 95% pure. The following compounds,α-6-mPEG₁-O-oxycodone (aka α-6-mPEG₁-O-hydroxycodone) (Compound C, n=1)431 mg of HCl salt 99% pure; and α-6-mPEG₂-O-oxycodone (akaα-6-mPEG₂-O-hydroxycodone) (Compound C, n=2) 454 mg HCl salt, 98% pure,were similarly prepared.

Example 18 In-Vivo Analgesic Assay Phenylquinone Writhing

An analgesic assay was used to determine whether exemplaryPEG-oligomer-opioid agonist conjugates belonging to the followingconjugate series: mPEG_(2-7,9)-O-morphine, mPEG_(3-7, 9)-O-codeine, andmPEG_(1-4,6, 7, 9)-O-hydroxycodone, are effective in reducing and/orpreventing visceral pain in mice.

The assay utilized CD-1 male mice (5-8 mice per group), each mouse beingapproximately 0.020-0.030 kg on the study day. Mice were treatedaccording to standard protocols. Mice were given a single “pretreatment”dose of a compound lacking covalent attachment of a water-soluble,non-peptidic oligomer (i.e., non-PEG oligomer-modified parent molecule),a corresponding version comprising the compound covalently attached to awater-soluble, non-peptidic oligomer (i.e., the conjugate), or controlsolution (IV, SC, IP or orally) thirty minutes prior to theadministration of the phenylquinone (PQ) solution. Each animal was givenan IP injection of an irritant (phenylquinone, PQ) that induces“writhing” which may include: contractions of the abdomen, twisting andturning of the trunk, arching of the back, and the extension of thehindlimbs. Each animal was given an IP injection of PQ (1 mg/kg PQ, 0.1mL/10 g bodyweight). After the injection, the animals were returned totheir observation enclosure and their behavior was observed.Contractions were counted between 35 and 45 minutes after the‘pretreatment”. The animals were used once. Each tested article wasdosed at a range between 0.1 and 100 mg/kg (n=5-10 animals/dose).

The results are shown in FIG. 4 (mPEG_(2-7,9)-O-morphine and control),FIG. 5 (mPEG_(1-4,6, 7, 9)-O-hydroxycodone and control), and FIG. 6(mPEG_(3-7, 9)-O-codeine and control). ED50 values are provided inTables 3A and 3B below.

TABLE 3A ED₅₀ values for mPEG_(n)-O-Morphine Series MORPHINE PEG 2 PEG 3PEG 4 PEG 5 PEG 6 PEG 7 PEG 9 ED₅₀ 0.3693 2.512 13.58 3.281 13.4 n/a n/an/a (mg/kg)

TABLE 3B ED₅₀ values for mPEG_(n)-O-HydroxyCodone Series OXYCODONE PEG 1PEG 2 PEG 3 PEG 4 PEG 6 PEG 7 PEG 9 ED₅₀ 0.6186 6.064 n/a n/a 17.31 n/an/a n/a (mg/kg)

Example 19 In-Vivo Analgesic Assay Hot Plate Latency Assay

A hot plate latency analgesic assay was used to determine whetherexemplary PEG-oligomer-opioid agonist conjugates belonging to thefollowing conjugate series: mPEG₁₋₅-O-morphine, mPEG₁₋₅-O-hydroxycodone,and mPEG_(2-5, 9)-O-codeine, are effective in reducing and/or preventingvisceral pain in mice.

The assay utilized CD-1 male mice (10 mice per group), each mouse beingapproximately 0.028-0.031 kg on the study day. Mice were treatedaccording to standard protocols. Mice were given a single “pretreatment”dose of a compound lacking covalent attachment of a water-soluble,non-peptidic oligomer (unmodified parent molecule), a correspondingversion comprising the compound covalently attached to a water-soluble,non-peptidic oligomer (i.e., the conjugate), or control solution (SC)thirty minutes prior to the hot plate test. The hot plate temperaturewas set at 55±1° C., calibrated with a surface thermometer beforecommencement of the experiment. Thirty minutes after “pretreatment”,each mouse was placed on the hot plate, and latency to lick a hindpawwas recorded to the nearest 0.1 second. If no lick occurred within 30seconds, the mouse was removed. Immediately after hot plate testing, atemperature probe was inserted 17 mm into the rectum, and bodytemperature was read to the nearest 0.1° C. when the meter stabilized(approximately 10 seconds). The animals were used once. Each testedarticle was dosed at a range between 0.3 and 30 mg/kg (n=5-10animals/dose).

Results are shown in FIG. 7 (hydroxycodone series), FIG. 8 (morphineseries) and FIG. 9 (codeine). Plots illustrate latency (time to lickhindpaw, in seconds) versus dose of compound administered in mg/kg.

Example 20 Pharmacokinetics of PEG_(oligo)-Opioid Compounds FollowingIntravenous (IV) and Oral (PO) Dosing in Male Sprague-Dawley Rats—StudyDesign

One seventy five (175) adult male Sprague-Dawley rats with indwellingjugular vein and carotid artery catheters (JVC/CAC) (Charles River Labs,Hollister, Calif.) were utilized for the study. There were 3 rats/group.All animals were food fasted overnight. Prior to dosing the rats wereweighed, the tails and cage cards were labeled for identification andthe doses were calculated. Anesthesia was induced and maintained with3.0-5.0% isoflurane. The JVC and CAC were externalized, flushed withHEP/saline (10 IU/mL HEP/mL saline), plugged, and labeled to identifythe jugular vein and carotid artery. The predose sample was collectedfrom the JVC. When all of the animals had recovered from anesthesia andthe predose samples were processed, the animals for intravenous groupwere were dosed, intravenously (IV) via the JVC using a 1 mL syringecontaining the appropriate test article, the dead volume of the catheterwas flushed with 0.9% saline to ensure the animals received the correctdose and oral group animals were treated orally via gavage.

Following a single IV dose, blood samples were collected at 0 (pre-dosecollected as described above), 2, 10, 30, 60, 90, 120, and 240 minutesand following oral dose, blood samples were collected 0 (pre-dosecollected as described above), 15, 30, 60, 120, 240 and 480 minutes viathe carotid artery catheter and processed as stated in the protocol.Following the last collection point, the animals were euthanized.

Bioanalytical analysis of the plasma samples was conducted usingLC-MS/MS methods.

Pharmacokinetic Analyses:

PK analysis was performed using WinNonlin (Version 5.2, Mountain View,Calif.-94014). Concentrations in plasma that were below LLOQ werereplaced with zeros prior to generating Tables and PK analysis. Thefollowing PK parameters were estimated using plasma concentration-timeprofile of each animal:

-   -   C₀ Extrapolated concentration to time “zero”    -   C_(max) Maximum (peak) concentration    -   AUC_(all) Area under the concentration-time from zero to time of        last concentration value    -   T_(1/2(Z)) Terminal elimination half-life    -   AUC_(inf) Area under the concentration-time from zero to time        infinity    -   T_(max) Time to reach maximum or peak concentration following        administration    -   CL Total body clearance    -   V_(z) Volume of distribution based on terminal phase    -   V_(ss) Volume of distribution at steady state    -   MRT_(last) Mean residence time to last observable concentration    -   F Bioavailability

Oral bioavailability was estimated using mean dose-normalized AUCalldata for the compounds where one of IV or PO groups with only reporteddata for <n=3/group.

Example 21 IV and PO Pharmacokinetics of mPEG_(n)-O-HydroxycodoneConjugates

A pharmacokinetic study was conducted in Sprague-Dawley rats asdescribed in Example 20 above. Compounds administered weremPEG_(n)-O-hydroxycodone conjugates where n=1, 2, 3, 4, 5, 6, 7, and 9,as well as the parent compound, oxycodone. The objective was todetermine the pharmacokinetics of the parent compound and its variousoligomer conjugates administered both intravenously and orally.

A summary of plasma PK parameters following IV (1 mg/kg) and PO (5mg/kg) delivery for oxycodone, mPEG₀-oxycodone, mPEG₁-O-hydroxycodone,mPEG₂-O-hydroxycodone, mPEG₃-O-hydroxycodone, mPEG₄-O-hydroxycodone,mPEG₅-O-hydroxycodone, mPEG₆-O-hydroxycodone, mPEG₇-O-hydroxycodone, andmPEG₉-O-hydroxycodone, are shown in the following tables, Tables 4 and5.

Based on the observed data (Table 4) for IV administration,mPEG₉-O-hydroxycodone appeared to achieve higher plasma concentrationwith a mean t_(1/2) value 3 times that of the corresponding mean t_(1/2)value observed after parent oxycodone was given.

FIG. 10 shows the mean plasma concentration-time profiles forIV-administered mPEGn-O-hydroxycodone compounds as described above, aswell as for oxycodone per se, when administered at a concentration of1.0 mg/kg.

Based on the observed data (Table 5) for oral administration,mPEG₅-O-hydroxycodone, mPEG₆-O-hydroxycodone, and mPEG₇-O-hydroxycodoneappeared to achieve higher mean exposure (approximately 3- to 8-fold) inplasma as compared to parent molecule, oxycodone.

FIG. 11 shows the mean plasma concentration-time profiles for themPEG_(n)-O-hydroxycodone compounds described above, as well as foroxycodone, when administered orally to rats at a concentration of 5.0mg/kg.

TABLE 4 Comparative PK Parameters of mPEG_(n)-O-hydroxycodone conjugatesgiven intravenously to rats (Mean ± SD) PEG- C_(max) T_(1/2)(z)AUC_(all) AUC_(inf) MRT_(last) CL V_(ss) length (ng/mL) min (min ·ng/mL) (min · ng/mL) min (mL/min/kg) (L/kg) 0 495 ± 56.0 47.0 ± 3.9912800 ± 1090 13000 ± 1070 37.0 ± 1.28 77.1 ± 6.26 3.17 ± 0.293 1 425 ±41.3 47.2 ± 6.37  9890 ± 1320 10100 ± 1440 38.7 ± 4.54  100 ± 13.4 4.31± 0.222 2 513 ± 48.8 44.6 ± 1.80 12000 ± 1610 12200 ± 1650 37.0 ± 2.6083.3 ± 10.8 3.36 ± 0.298 3 746 ± 2.08 48.5 ± 7.83 13800 ± 1050 14000 ±1010 32.5 ± 1.92 71.7 ± 4.99 2.62 ± 0.206 4 537 ± 31.0 43.6 ± 3.27 11500± 783  11600 ± 827  35.6 ± 2.88 86.5 ± 6.36 3.34 ± 0.113 5 622 ± 39.762.1 ± 3.85 16900 ± 1800 17700 ± 1990 46.2 ± 1.86 57.0 ± 6.07 3.30 ±0.184 6 445 ± 83.6 62.2 ± 5.17 12600 ± 2370 13100 ± 2390 47.7 ± 1.4177.9 ± 14.4 4.68 ± 0.938 7 489 ± 26.5 87.0 ± 3.25 14300 ± 583  15800 ±728   54.3 ± 0.372 63.3 ± 2.99 5.31 ± 0.139 9 955 ± 149   143 ± 14.316600 ± 2190 21000 ± 4230 52.7 ± 4.04 48.9 ± 9.41 6.35 ± 0.349

TABLE 5 Comparative PK Parameters of mPEG_(n)-O-hydroxycodone conjugatesgiven orally to Sprague Dawley rats (Mean ± SD) PEG- C_(max) T_(1/2(z))AUC_(all) AUC_(inf) T_(max)* MRT_(last) length (ng/mL) min (min · ng/mL)(min · ng/mL) min min F % 0 25.5 ± 1.86 NC 4520 ± 1660 NC 15.0 179 ±17.4 7.1 1 14.3 ± 6.43 57.7* 1050 ± 205  1150* 15.0 66.8 ± 23.8  2.1 299.4 ± 47.3 48.5 ± 12.0 5910 ± 2690 5830 ± 2600 15.0 55.4 ± 14.7  9.4 344.5 ± 29.4 65.6* 3620 ± 1910 4210* 15.0 84.7 ± 17.0  5.3 4 55.8 ± 4.6970.3* 6340 ± 1810 5280* 15.0 96.6 ± 33.6  11.0 5  178 ± 14.7 75.8 ± 1.0832800 ± 2020  33300 ± 2090  15.0 124 ± 4.84 37.6 6  171 ± 76.6 85.4 ±7.83 35100 ± 10100 36200 ± 10200 120 154 ± 6.46 55.3 7  114 ± 38.0  115± 29.2 20400 ± 3670  22200 ± 2900  120 178 ± 6.09 28.1 9 27.6 ± 19.6 106(n = 1) 7620 ± 4510 13500 (n = 1) 120 203 ± 43.8 9.2 *n = 2, NC: Notcalculated. Tmax is reported as median value.

To summarize the results, intravenous administration of PEGylatedhydroxycodone with varying oligomeric PEG-lengths (PEG1 to PEG9)resulted in variable plasma concentrations and exposures as compared tooxycodone. PEGs with chain lengths 3, 5, 7 and 9 showed higher meanexposure (AUC) while PEG6 showed comparable mean exposure (AUC) and PEGswith chain lengths 1, 2 or 4 showed slightly lower mean exposure (AUC).The compounds having a PEG length greater than 5 showed trends of lowerclearance, higher volume of distribution at steady state, increase inelimination half life values, with increasing PEG length.

Oral administration of PEGylated hydroxycodone with varying oligomericPEG-lengths (PEG1 to PEG9) resulted in improvement in plasma exposurewith the exception of hydroxycodone covalently attached to PEG1 and toPEG3. Oral bioavailability was highest for hydroxycodone covalentlyattached to mPEG6, 55.3%) followed by mPEG5-hydroxycodone andmPEG7-hydroxycodone with 37.6% and 28.1%, respectively. The eliminationhalf-life values showed a trend of increasing with increase inPEG-length.

Example 22 IV and PO Pharmacokinetics of mPEG_(n)-O-Morphine Conjugates

A pharmacokinetic study was conducted in Sprague-Dawley rats asdescribed in Example 20 above. Compounds administered weremPEG_(n)-O-morphine conjugates where n=1, 2, 3, 4, 5, 6, 7, and 9, aswell as the parent compound, morphine. The objective was to determinethe pharmacokinetics of the parent compound and its various oligomerconjugates administered both intravenously and orally.

A summary of plasma PK parameters following IV (1 mg/kg) and PO (5mg/kg) routes for morphine, mPEG₁-O-morphine, mPEG₂-O-morphine,mPEG₃-O-morphine, mPEG₄-O-morphine, mPEG₅-O-morphine, mPEG₆-O-morphine,mPEG₇-O-morphine, mPEG₉-O-morphine, are shown in Table 6 and Table 7,respectively.

For the intravenous group: FIG. 12 shows the mean plasmaconcentration-time profiles for the above mPEG_(n)-O-morphine conjugatesafter 1.0 mg/kg intravenous administration to rats. There appeared to beone outlier datum in each animal that are inconsistent with plasmaprofiles of mPEG₂-O-morphine, and were excluded from the PK analysis.

Based on the observed data (Table 6), mPEG₉-O-morphine appeared toachieve higher plasma concentration with a mean t_(1/2) value 4 timesthat of the corresponding t_(1/2) value observed after parent morphinewas given.

TABLE 6 Comparative PK Parameters of mPEG_(n)-O-morphine Conjugatesgiven intravenously to rats PEG- C_(max) T_(1/2(z)) AUC_(all) AUC_(inf)MRT_(last) CL V_(ss) Length (ng/mL) min (min · ng/mL) (min · ng/mL) min(mL/min/kg) (L/kg) 0 132 ± 5.86 51.1 ± 20.8 2730 ± 276 2760 ± 218 28.5 ±6.79  364 ± 27.5 14.9 ± 4.0  1 483 ± 37.1 40.0 ± 2.58 11400 ± 1230 11500± 1260 29.8 ± 5.05 87.8 ± 9.40 2.75 ± 0.236 2 378 ± 48.8 38.1 ± 8.037510 ± 106 7410 ± 404 26.4 ± 5.90  135 ± 7.60  4.2 ± 0.270 3 483 ± 81.045.0 ± 2.73 12700 ± 1950 12900 ± 1990 39.3 ± 1.69 78.5 ± 11.8 3.43 ±0.616 4 622 ± 72.5 52.9 ± 6.50 14600 ± 1140 15000 ± 1270  40.1 ± 0.96267.1 ± 5.58 3.17 ± 0.168 5 514 ± 38.6  68.4 ± 0.826 13200 ± 998  14000 ±1050 49.7 ± 1.20 71.6 ± 5.17 4.74 ± 0.347 6 805 ± 30.6 93.7 ± 17.1 19000± 1430 21600 ± 2060 56.2 ± 3.84 46.6 ± 4.67 4.39 ± 0.630 7 1110 ± 123   111 ± 32.9 18100 ± 956  21200 ± 1990 49.6 ± 5.20 47.4 ± 4.21 4.76 ±0.997 9 1840 ± 123    204 ± 28.3 23300 ± 1460 29000 ± 3240 34.2 ± 2.7234.7 ± 3.64 4.52 ± 0.473

For the oral group, FIG. 13 shows the mean plasma concentration-timeprofiles for the above described mPEG_(n)-O-morphine conjugates afterthe oral administration (5.0 mg/kg) to rats.

Based on the observed data (Table 7), mPEG₄-O-morphine appeared toachieve highest plasma concentrations among the conjugates tested ascompared to parent molecule, morphine.

TABLE 7 Comparative PK Parameters of mPEG_(n)-O-morphine conjugatesgiven orally to Sprague Dawley rats (Mean ± SD) PEG- C_(max) T_(1/2(z))AUC_(all) AUC_(inf) T_(max) MRT_(last) Length (ng/mL) min (min · ng/mL)(min · ng/mL) min min F % 0 29.8 ± 7.78 144 ± 32.1 5510 ± 667 7230 ±897  15.0 194 ± 22.0 40.4^(¥) 2 3.84* 104* 448*  778* 15.0* 60.7* 0.15 330.3 ± 4.42 377*  4250 ± 2140 8370* 15.0 151 ± 69.4 9.0 4 87.1 ± 53.6191 ± 104  15600 ± 7690 18200 ± 10300 30.0 149 ± 26.7 22.1 5 35.6 ± 19.8247*  9190 ± 5650 17400*  120 205 ± 26.2 13.9 6 42.8 ± 31.2 121*  8290 ±4970 10800*  120 177 ± 29.4 8.7 7  9.38 ± 0.883 236* 2210 ± 221 2720*60.0 187 ± 32.0 2.4 9 7.15 ± 3.34 363* 1360 ± 311 2270* 15.0 166 ± 26.01.2 No PK parameters were not reported for mPEG₁-morphine because allthe concentrations were <LLOQ. *n = 2.

In summary, for the IV data, administration of oligomeric PEGylatedmorphine with varying PEG-lengths (PEG1 to PEG9) resulted in higherplasma concentrations and exposure (AUC) as compared to morphine per se.There was a clear trend of increase in mean AUC with increase inPEG-length of 5 onwards, with 10-fold higher mean AUC for thePEG9-morphine compound as compared to non-conjugated morphine. The meanhalf-life and mean residence time also increased with increase inPEG-length. The lower mean clearance values were consistent withobserved higher mean AUC values.

Mean volume of distribution estimated for steady state, immediatelydecreased by 5-fold with the introduction of single PEG, and reached aconstant value at PEG-length 5. Overall, PEGylation appeared to increasethe elimination t_(1/2) and lower the tissue distribution of morphine.

Based upon the oral data, administration of PEGylated morphineconjugates with varying PEG-lengths (PEG1 to PEG9) resulted in areduction in oral bioavailability compared to morphine. The reduction inbioavailability appeared to be related to the absorption componentrather than metabolism component for these PEG-conjugates. Among thePEG-conjugates, the conjugate with PEG-length 4 showed maximum F-value(22.1%) while conjugates with shorter or longer PEG-length showed aclear trend of loss in absorption.

In this study, morphine F % value was 3-fold higher than literaturevalue of 15% at 7.5 mg/kg (J. Pharmacokinet. Biopharm. 1978, 6:505-19).The reasons for this higher exposure are not known.

Example 23 IV and PO Pharmacokinetics of mPEG_(n)-O-Codeine Conjugates

A pharmacokinetic study was conducted in Sprague-Dawley rats asdescribed in Example 20 above. Compounds administered weremPEG_(n)-O-codeine conjugates where n=1, 2, 3, 4, 5, 6, 7, and 9, aswell as the parent compound, codeine (n=0). The objective was todetermine the pharmacokinetics of the parent compound, i.e., codeine,and its various oligomer conjugates administered both intravenously andorally.

A summary of plasma PK parameters following IV (1 mg/kg) and PO (5mg/kg) routes for codeine, mPEG₁-O-codeine, mPEG₂-O-codeine,mPEG₃-O-codeine, mPEG₄-O-codeine, mPEG₅-O-codeine, mPEG₆-O-codeine,mPEG₇-O-codeine, mPEG₉-O-codeine, are shown in Table 8 and Table 9,respectively.

For the IV group: FIG. 14 shows the mean plasma-concentration-timeprofiles for parent molecule, codeine, as well as for themPEG_(n)-O-codeine conjugates described above, after intravenousadministration.

Based on the observed data (Table 8), mPEG₆-O-codeine appeared toachieve higher plasma concentrations among the tested conjugates with amean t_(1/2) value approximately 2.5 times that of the correspondingt_(1/2) value observed following administration of the parent molecule,codeine.

TABLE 8 Comparative PK Parameters of Codeine and its Oligomeric PEGConjugates Admnistered Intravenously to Rats PEG- C_(max) T_(1/2(z))AUC_(all) AUC_(inf) MRT_(last) CL V_(ss) Length (ng/mL) min (min ·ng/mL) (min · ng/mL) min (mL/min/kg) (L/kg) 0 469 ± 20.4 42.1 ± 3.1511000 ± 1600 11400 ± 2070 40.2 ± 9.08 89.7 ± 15.3 4.14 ± 0.700 1 723 ±31.2 42.1 ± 4.84 15500 ± 2020 15700 ± 2130 32.2 ± 4.59 64.6 ± 8.75 2.22± 0.899 2 685 ± 41.0 35.3 ± 2.78 14500 ± 1590 14600 ± 1590 31.5 ± 2.9669.0 ± 7.57 2.25 ± 0.166 3 732 ± 27.1 39.4 ± 1.49 17300 ± 1520 17400 ±1550 33.8 ± 2.40 57.7 ± 4.89 2.07 ± 0.127 4 746 ± 70.0 57.1 ± 43.8 15200± 2160 15400 ± 2240 27.5 ± 4.55 65.9 ± 10.4 2.30 ± 0.720 5 533 ± 38.942.7 ± 3.56 11500 ± 878  11700 ± 913  31.8 ± 1.53 86.2 ± 7.04 2.95 ±0.157 6 1780 ± 149   58.0 ± 4.79 45600 ± 2020 47100 ± 2000 41.7 ± 3.08 21.3 ± 0.876 1.08 ± 0.143 7 443 ± 43.3 74.5 ± 5.76 12700 ± 481  13700 ±320  50.7 ± 2.07 73.1 ± 1.73 5.20 ± 0.596 9 730 ± 68.0  109 ± 1.80 17800± 2310 20800 ± 2840 57.2 ± 2.46 48.6 ± 6.74 5.18 ± 0.538 Tmax isreported as median value. *n = 2.

For the oral group, FIG. 15 shows the mean plasma concentration-timeprofiles for parent molecule, codeine, versus mPEG_(n)-codeineconjugates after oral administration to rats (5.0 mg/kg).

Based on the observed data (Table 9), the PEG-6 compound, mPEG₆-codeine,appeared to achieve highest plasma concentrations (52 times higher meanAUCall) among the tested conjugates as parent molecule, codeine.

TABLE 9 Comparative PK Parameters of Codeine and VariousmPEG_(n)-Codeine Conjugates given Orally to Sprague Dawley Rats (Mean ±SD) PEG- C_(max) T_(1/2(z)) AUC_(all) AUC_(inf) T_(max) MRT_(last)Length (ng/mL) min (min · ng/mL) (min · ng/mL) min min F % 0 6.24 ± 2.5180.8^(# )  328 ± 216   431^(#) 15.0 33.2 ± 12.9 0.60 2  3.47 ± 0.60697.6 ± 28.4   351 ± 195 419 ± 226 15.0 62.0 ± 27.4 0.57 3 25.0 ± 6.59125 ± 64.6 1920 ± 245 2080 ± 498  15.0 71.0 ± 9.16 2.39 4 31.1 ± 13.1118 ± 60.0 2530 ± 682 2670 ± 870  15.0 83.8 ± 22.5 3.47 5 48.7 ± 10.8125 ± 63.7 5510 ± 963 5890 ± 1470 15.0  108 ± 35.4 10.1 6  617 ± 56.4126 ± 54.1  70500 ± 12300 74500 ± 10000 15.0  119 ± 11.1 31.6 7 76.6 ±12.8 97.6* 17100 ± 4220 16000* 120  171 ± 21.7 26.9 9 31.5 ± 8.43 143*   7320 ± 3330  6840* 15.0  179 ± 21.6 8.22 No PK parameters were notreported for NKT-10479 because the concentrations were LLOQ. ^(#)n = 1,*n = 2. T_(max) is reported as median value.

In summary, for the IV data, PEGylation of codeine with varyingoligomeric PEG-lengths (PEG1 to PEG9) improved exposure (mean AUC) onlyslightly and moderate improvement (approximately 4-fold) was observedfor the PEG-6 conjugate. Both clearance and volume of distributiondecreased for this PEG-conjugate by 4-fold. Conjugates with PEG-lengths7 and 9 showed longer mean t_(1/2) values, however, mean clearance andmean volume of distribution (Vss) were decreased for both for both thePEG7- and PEG9-codeine conjugates.

For the oral data, oral bioavailability for codeine is very low(F=0.52%). Oral bioavailability appeared to increase with increase inPEG-length from 2 onwards, reaching maximum with 32% bioavailability forthe codeine conjugate with PEG-length 6, decreasing thereafter. Ingeneral, mean t₁₁₂ and mean residence values increased with PEG-length.There was no difference in time to reach peak concentrations (Tmax=15min) amongst all the compounds tested, suggesting that absorption wasrapid and the absorption rate was not altered.

Example 24 In-Vitro Binding of mPEG_(n)-O-Opioid Conjugates to OpioidReceptors

The binding affinities of the various PEG-opioid conjugates(mPEG_(n)-O-morphine, mPEG_(n)-O-codeine, and mPEG_(n)-O-hydroxycodone)was measured in vitro in membrane preparations prepared from CHO cellsthat heterologously express the cloned human mu, kappa or delta opioidreceptors. Radioligand displacement was measured using scintillationproximity assays (SPA).

Briefly, serial dilutions of the test compounds were placed in a 96-wellplate to which were added SPA beads, membrane and radioligand. The assayconditions for each opioid receptor subtype are described in Table 10below. The plates were incubated for 8 hours-overnight at roomtemperature, spun at 1000 rpm to pellet the SPA beads, and radioactivitywas measured using the TopCount® microplate Scintillation counter.Specific binding at each concentration of test compound was calculatedby subtracting the non-specific binding measured in the presence ofexcess cold ligand. IC₅₀ values were obtained by non-linear regressionof specific binding versus concentration curves and Ki values werecalculated using Kd values that were experimentally pre-determined foreach lot of membrane preparations.

TABLE 10 Assay conditions for opioid receptor binding assays.EXPERIMENTAL MU OPIOID KAPPA OPIOID DELTA OPIOID VARIABLE RECEPTORRECEPTOR RECEPTOR SPA beads PVT-WGA PEI Type A PVT-WGA (GE PVT-WGA PEIType B (GE Healthcare, Cat. # Healthcare, Cat. (GE Healthcare, Cat.RPNQ0003) #RPNQ0001) #RPNQ0004) Radioligand; DAMGO, [Tyrosyl-3,5-U-69,593, [Phenyl-3,4- Naltrindole, [5′,7′-3H]- Concentration3H(N)]-(Perkin Elmer, 3H]-(Perkin Elmer, Cat. (Perkin Elmer, Cat. Cat. #NET-902); 6 nM #NET-952); 10 nM #NET-1065); 3 nM Non-specific CTAPnor-Binaltorphimine SNC80 binding control (nor-BNI) Buffer 50 mMTris-HCl, pH 7.5 50 mM Tris-HCl, pH 7.5 50 mM Tris-HCl, pH 7.5 5 mMMgCl2; 5 mM MgCl2 5 mM MgCl2 1 mM EDTA Receptor and Recombinant human muRecombinant human Recombinant human delta source opioid receptorexpressed kappa opioid receptor opioid receptor expressed in CHO-K1 hostcell expressed in Chem-1 host in Chem-1 host cell membranes (PerkinElmer, cell membranes membranes (Millipore, Cat. #ES-542-M) (Millipore,Cat. Cat. #HTS100M). #HTS095M)

The binding affinities of the oligomeric PEG conjugates of morphine,codeine and hydroxycodone are shown in Table 11. Overall, all of theconjugates displayed measurable binding to the mu-opioid receptor,consistent with the known pharmacology of the parent molecules. For agiven PEG size, the rank order of mu-opioid binding affinity wasPEG-morphine>PEG-hydroxycodone>PEG-codeine. Increasing PEG size resultedin a progressive decrease in the binding affinity of all PEG conjugatesto the mu opioid receptor compared to unconjugated parent molecule.However, the PEG-morphine conjugates still retained a high bindingaffinity that was within 15× that of parent morphine. The mu-opioidbinding affinities of PEG-hydroxycodones were 20-50 fold lower thanthose of the PEG-morphine conjugates. Codeine and its PEG conjugatesbound with very low affinity to the mu opioid receptor. PEG-morphineconjugates also bound to the kappa and delta opioid receptors; the rankorder of selectivity was mu>kappa>delta. Binding affinities of codeineand hydroxycodone conjugates to the kappa and delta opioid receptorswere significantly lower than that at the mu-opioid receptor.

TABLE 11 Binding affinities of the PEG-opioid conjugates to opioidreceptors. KI (NM) Mu Kappa Delta opioid opioid opioid COMPOUND receptorreceptor receptor Morphine 8.44 118.38 4,297 α-6-mPEG₁-O-Morphine 15.72444.54 2,723 α-6-mPEG₂-O-Morphine 21.97 404.33 2,601α-6-mPEG₃-O-Morphine 50.66 575.98 6,176 α-6-mPEG₄-O-Morphine 23.11438.88 3,358 α-6-mPEG₅-O-Morphine 39.40 557.54 2,763α-6-mPEG₆-O-Morphine 72.98 773.56 2,595 α-6-mPEG₇-O-Morphine 56.86669.56 2,587 α-6-mPEG₉-O-Morphine 111.05 1253.71 5,783 Oxycodone 133.48N/A N/A α-6-mPEG₁-O-Hydroxycodone 653.90 N/A N/Aα-6-mPEG₂-O-Hydroxycodone 631.76 N/A N/A α-6-mPEG₃-O-Hydroxycodone775.19 N/A N/A α-6-mPEG₄-O-Hydroxycodone 892.70 N/A N/Aα-6-mPEG₅-O-Hydroxycodone 1862.14 N/A N/A α-6-mPEG₆-O-Hydroxycodone1898.30 N/A N/A α-6-mPEG₇-O-Hydroxycodone 1607.19 N/A N/Aα-6-mPEG₉-O-Hydroxycodone 3616.60 N/A N/A Codeine 1,953 28,067 N/Aα-6-mPEG₁-O-Codeine 1821.51 54669.89 N/A α-6-mPEG₂-O-Codeine 1383.0722603.05 N/A α-6-mPEG₃-O-Codeine 4260.21 36539.78 N/Aα-6-mPEG₄-O-Codeine 2891.36 96978.61 N/A α-6-mPEG₅-O-Codeine 2427.1359138.22 N/A α-6-mPEG₆-O-Codeine 14202.77 >160,000 N/Aα-6-mPEG₇-O-Codeine 9963.93 108317.50 N/A α-6-mPEG₉-O-Codeine 9975.8472246.23 N/A N/A indicates that Ki values could not be calculated sincea 50% inhibition of binding was not achieved at the highestconcentration of compound tested.

Example 25 In-Vitro Efficacy of mPEG_(n)-O-Opioid Conjugates to InhibitcAMP Formation

The efficacy of the various PEG-opioid conjugates was measured by theirability to inhibit cAMP formation following receptor activation. Studieswere conducted in CHO cells heterologously expressing the cloned humanmu, kappa or delta opioid receptors. cAMP was measured using a cAMPHiRange homogenous time-resolved fluorescence assay (HTRF Assay), thatis based on a competitive immunoassay principle (Cisbio, Cat.#62AM6PEC).

Briefly, suspensions of cells expressing either the mu, kappa or deltaopioid receptors were prepared in buffer containing 0.5 mMisobutyl-methyl xanthine (IBMX). Cells were incubated with varyingconcentrations of PEG-opioid conjugates and 3 μM forskolin for 30minutes at room temperature. cAMP was detected following a two-stepassay protocol per the manufacturer's instructions and time resolvedfluorescence was measured with the following settings: 330 nmexcitation; 620 nm and 665 nm emission; 380 nm dichroic mirror. The 665nm/620 nm ratio is expressed as Delta F % and test compound-related datais expressed as a percentage of average maximum response in wellswithout forskolin. EC₅₀ values were calculated for each compound from asigmoidal dose-response plot of concentrations versus maximum response.To determine if the compounds behaved as full or partial agonists in thesystem, the maximal response at the highest tested concentrations ofcompounds were compared to that produced by a known full agonist.

The EC₅₀ values for inhibition of cAMP formation in whole cells areshown in Table 12. Oligomeric PEG conjugates of morphine, codeine andhydroxycodone were full agonists at the mu opioid receptor, albeit withvarying efficacies. Morphine and its conjugates were the most potent ofthe three series of opioids tested, while the PEG hydroxycodone and PEGcodeine conjugates displayed significantly lower efficacies. Aprogressive decrease in the efficacy of the PEG-morphine conjugates wasobserved with increasing PEG size, however the conjugates retainedmu-agonist potency to within 40× of parent. In contrast to the effect atthe mu opioid receptor, morphine and PEG-morphine conjugates behaved asweak partial agonists at the kappa opioid receptor, producing 47-87% ofthe maximal possible response. EC₅₀ values could not be calculated forthe codeine and hydroxycodone conjugates at the kappa and delta opioidreceptors since complete dose-response curves could not be generatedwith the range of concentrations tested (up to 500 μM).

Overall, the results of the receptor binding and functional activityindicate that the PEG-opioids are mu agonists in vitro.

TABLE 12 In vitro efficacies of PEG-opioid conjugates MU OPIOID KAPPAOPIOID RECEPTOR RECEPTOR DELTA EC₅₀, % max EC₅₀, % max OPIOID COMPOUNDnM effect nM effect RECEPTOR Morphine 28.5 102 624 69 N/Aα-6-mPEG₁-O-Morphine 85.0 91 1,189 81 N/A α-6-mPEG₂-O-Morphine 93.3 91641 87 N/A α-6-mPEG₃-O-Morphine 270 100 4,198 82 N/Aα-6-mPEG₄-O-Morphine 128 100 3,092 77 N/A α-6-mPEG₅-O-Morphine 157 952,295 71 N/A α-6-mPEG₆-O-Morphine 415 98 3,933 62 N/Aα-6-mPEG₇-O-Morphine 508 90 4,237 57 N/A α-6-mPEG₉-O-Morphine 1,061 874,417 47 N/A Oxycodone 478 95 N/A N/A N/A Hydroxycodone 3,162 N/A N/Aα-6-mPEG₁-O-Hydroxycodone 3,841 102 N/A N/A N/Aα-6-mPEG₂-O-Hydroxycodone 5,005 101 N/A N/A N/Aα-6-mPEG₃-O-Hydroxycodone 2,827 108 N/A N/A N/Aα-6-mPEG₄-O-Hydroxycodone 3,715 109 N/A N/A N/Aα-6-mPEG₅-O-Hydroxycodone 5,037 108 N/A N/A N/Aα-6-mPEG₆-O-Hydroxycodone 12,519 102 N/A N/A N/Aα-6-mPEG₇-O-Hydroxycodone 7,448 101 N/A N/A N/Aα-6-mPEG₉-O-Hydroxycodone 17,948 95 N/A N/A N/A Codeine 10,418 81 N/A  3N/A α-6-mPEG₁-O-Codeine 8,574 80 N/A 51 N/A α-6-mPEG₂-O-Codeine 5,145 7540,103 59 N/A α-6-mPEG₃-O-Codeine 19,740 91 N/A 49 N/Aα-6-mPEG₄-O-Codeine 22,083 99 N/A 61 N/A α-6-mPEG₅-O-Codeine 23,235 95N/A 60 N/A α-6-mPEG₆-O-Codeine 97,381 80 N/A 21 N/A α-6-mPEG₇-O-Codeine44,729 75 N/A 48 N/A α-6-mPEG₉-O-Codeine 48,242 80 N/A 61 N/A

Example 26 Brain:Plasma Ratios of mPEG_(n)-O-Opioid Conjugates

The ability of oligomeric mPEG-O-morphine, mPEG-O-codeine andmPEG-O-hydroxycodone conjugates to cross the blood brain barrier (BBB)and enter the CNS (central nervous system) was assessed by measuring thebrain:plasma ratio in rats subsequent to IV administration.

Briefly, groups of 3 rats were injected intravenously (i.v) with 5 mg/kgeach of morphine, mPEG_(n)-O-morphine conjugate, codeine andm-PEG_(n)-O-codeine conjugates. PEG-2, 3 and 4-oxycodone conjugates wereadministered at 10 mg/kg i.v. and oxycodone and the other PEG sizes ofoxycodone conjugates were administered at 1 mg/kg (i.v). The doses ofthe oxycodone conjugates had to be adjusted to allow for the detectionof sufficient concentrations in brain tissue. Atenolol, which does notcross the BBB, was used as a measure of vascular contamination of thebrain tissue and was administered at a concentration of 10 mg/kg to aseparate group of rats. An hour following injection, the animals weresacrificed and plasma and the brain were collected and frozenimmediately. Following tissue and plasma extractions, concentrations ofthe compounds in brain and plasma were measured using LC-MS/MS. Thebrain:plasma ratio was calculated as the ratio of measuredconcentrations in the brain and plasma. The results are shown in FIGS.16A-C.

FIGS. 16A, 16B, and 16C show the brain:plasma ratios of variousoligomeric mPEG_(n)-O-morphine, mPEG_(n)-O-codeine, andPEG_(n)-O-hydroxycodone conjugates, respectively. The brain:plasma ratioof atenolol is shown in each figure to provide a basis for comparison.PEG-conjugation results in a decrease in the brain:plasma ratio of allconjugates compared to their respective unconjugated parent molecule,which in the case of hydroxycodone is oxycodone. Only PEG-1-morphinedisplayed a greater brain:plasma ratio than its parent, morphine.

Example 27 Time-Course of Brain and Plasma Concentrations of VariousExemplary mPEG_(n)-O-Opioid Conjugates

Experiments were conducted to determine the concentrations of variousoligomeric PEG-opioid conjugates in brain and plasma over time followingIV administration.

The experimental methodology and concentrations used were the same asthose used for the single time point experiments described in Example26, however, the brains and plasma were harvested at various differingtime points.

All PEG-hydroxycodone conjugates were administered at 10 mg/kg iv, whilethe oxycodone parent was administered at 1 mg/kg iv. The data for thebrain and plasma concentrations versus time for the various PEG-opioidconjugates administered is shown in FIGS. 17A-H (morphine series), FIGS.18A-H (codeine series), and FIGS. 19A-H (oxycodone/hydroxycodoneseries).

The data demonstrate that the maximal increase in brain concentrationsfor all parent molecules and oligomeric PEG-conjugates occurs at theearliest time point, i.e., 10 minutes following iv injection. PEGconjugation results in a significant reduction in the brainconcentrations and with the larger PEG conjugates (≧PEG-4), the brainconcentrations remain relatively low and steady over time.

1-33. (canceled)
 34. A method of producing a compound of Formula C:

comprising reacting α-6-hydroxycodone with an activated sulfonate esterof methoxy-terminated poly(ethylene glycol) to form the compound ofFormula C:


35. The method of claim 34, wherein the α-6-hydroxycodone is produced byreduction of oxycodone.
 36. The method of claim 34, wherein the reactionis carried out at a temperature from 60° C. to 80° C.
 37. The method ofclaim 34, wherein the reaction is carried out in a mixture of tolueneand DMF.
 38. The method of claim 34, wherein the reaction is carried outin the presence of NaH.